Cell-targeting molecule comprising a mutant human carboxypeptidase A

ABSTRACT

Conjugates of a cell targetting molecule and a mutant human carboxypeptidase A enzyme are provided. Suitable targetting molecules include antibodies, hormones, ligands, cytokines, antigens, oligonucleotides and peptidomimetics. Enzymes comprising a mutant human carboxypeptidase A enzyme are also provided.

This application is filed pursuant to 35 U.S.C. §371 as a United States National Phase Application of International Application No. PCT/GB94/02483 filed 11 Nov. 11, 1994 which claims priority from GB 9323429.2 filed Nov. 12, 1993.

FIELD OF THE INVENTION

The present invention relates to improvements in targetted enzyme prodrug therapy including antibody-directed enzyme prodrug therapy (ADEPT), it particularly relates to novel enzymes and prodrugs for use in ADEPT.

In the therapy of certain conditions it is preferable that a drug be delivered onto to a particular cellular subpopulation. For example the use of drugs in the treatment of cancer is limited by the inability of the cytotoxic drug to differentiate between cells exhibiting normal cell division and those exhibiting neoplastic division. Hence the therapy is not targeted to a clinically acceptable extent and healthy cells are exposed to cytotoxin. Conjugation of a drug to an antibody, preferably a monoclonal antibody (mAb), allows the targeting of the drug to a particular cellular subpopulation expressing the antigenic determinant to which the targetting antibody binds. However, factors, such as the inability of the conjugate to penetrate the relevant tissue, poor release of the drug from the antigen bound conjugate and the limitation placed on the amount of drug which can be delivered by the number of available antibody-binding sites, have limited the effectiveness of this approach.

BACKGROUND

Avoidance of such limitations led to the concept of targeting conjugates of antibodies and enzymes capable of converting relatively non-cytotoxic `prodrugs` into low molecular weight cytotoxins at the antibody-binding site. This general concept was disclosed by Rose in European Patent application 84302218.7. Bagshawe and collaborators have referred to the concept as ADEPT, Antibody Directed Enzyme Prodrug Therapy. (Bagshawe K. D. et.al., Br. J. Cancer [1987] 56, 531-532, Bagshawe K. D. et al., Br. J. Cancer [1988] 50, 700-703 and WO 90/10140). In this way one conjugate could generate a proportionately larger amount of drug at the target site by repeated rounds of enzymatic catalysis of prodrug activation.

EP 382 411 describes ADEPT wherein a prodrug may be converted to a cytotoxic agent by enzymes including beta-lactamases isolated from various micro-organisms, L-pyroglutamate aminopeptidase, beta-galactosidase, D-amino acid peptidase, isoenzymes of alkaline phosphatase and various carboxypeptidases.

WO 91/11201 describes ADEPT wherein a prodrug may be enzymatically cleaved to generate cyanide by β-glycosidases or β-glucosidases, generally of plant origin.

EP 302 473 describes ADEPT wherein the enzyme alkaline phosphatase may be used to cleave novel prodrugs of mitomycin, penicillin V amidase may be used to cleave novel prodrugs of adriamycin, or cytosine deaminase may be used with the prodrug 5-fluorocytosine.

EP 484 870 describes ADEPT wherein β-lactamase may be used to activate a cephalosporin prodrug to yield a cytotoxic nitrogen mustard.

WO 88/07378 describes ADEPT wherein benzoic acid nitrogen mustard glutamides may be converted to the nitrogen mustard under the action of carboxypeptidases.

Vitols, K. S., et al, Pteridines 3, [1992], 125-126, discloses a number of MTX-amino acid prodrugs which may be activated by carboxypeptidase-mAb conjugates as part of ADEPT.

WO 91/09134 discloses bispecific hybrid mAbs for use in ADEPT wherein the mAb has specificities against both human cancer cell antigens and a prodrug-activating enzyme.

All previously disclosed methods of ADEPT may be divided into two categories: those which employ human enzymes and those which employ non-human (eg. bacterial) enzymes to activate the relevant prodrug. Both strategies retain inherent problems which limit their potential to provide effective therapy. Use of a human enzyme results in instability of the associated prodrug in vivo, as it may be activated at sites distant to the target site where endogenous human enzymatic activity may occur naturally. Clearly this will also have the highly undesirable effect of generating potentially cytotoxic compounds in non-targetted areas of the body with possibly lethal consequences. The use of a non-human enzyme permits the associated prodrug, which is only activated by the non-human enzymatic activity, to avoid activation by endogenous enzymes and so remain stable in vivo until converted to drug at the target site. However, such a non-human enzyme may elicit an immune response when introduced in vivo and antibodies generated to the enzyme will limit or destroy its ability to activate the prodrug.

WO 90/07929 identifies the desideratum of a non-endogenous catalytic activity being provided by a non-immunogenic enzyme but teaches only that this may be achieved by the use of "genetically conserved" enzymes or those from a "genetically similar species". However it is not taught how this may be accomplished.

It has now been found that the apparent conflict arising from the need to provide a prodrug which is stable in vivo and yet activated by a non-immunogenic enzyme may be resolved by generation of a mutant mammalian enzyme which retains catalytic activity but possesses a novel substrate specificity. The associated prodrug may be activated by the catalytic activity of the mutant enzyme but since the substrate specificity of the mutant enzyme is not a naturally occurring one, the prodrug remains stable in vivo until converted to drug at the target site. The ability to use a non-immunogenic enzyme according to the present invention provides the further advantage that repeated rounds of therapy may be administered. This is in contrast to known processes for ADEPT, in which the initial introduction of the enzyme to the system elicits an immune response which effectively precludes further treatment with the same enzyme as this will be removed from the body by an immune reaction `primed` during the first round of therapy.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a method of targetting a chemotherapeutic agent to a specific cell-type comprising the administration to a mammal of (i) an effective amount of a conjugate of a cell-type specific targetting molecule with a mutant mammalian enzyme capable of catalysing a functionally inactive precursor of a chemotherapeutic agent to its active form and (ii) an effective amount of the functionally inactive precursor of the chemotherapeutic agent which is refractory to endogenous catalysis to the chemotherapeutic agent.

Endogenous catalysis implies conversion of functionally inactive precursor of a chemotherapeutic agent to that chemotherapeutic agent by enzymes naturally present in vivo. Clearly, conversion occurring at the target site, catalysed by the targetted mutant mammalian enzyme, is not considered to be endogenous catalysis.

A further aspect of the present invention provides a method of treatment of a mammal requiring therapy for any of the conditions hereinafter described comprising the administration to the mammal of an effective amount of a cell-type specific targetting molecule conjugated with a mutant mammalian enzyme which is capable of catalysing a functionally inactive precursor of a drug to its active form in combination with an effective amount of a functionally inactive precursor of the drug which is refractory to endogenous catalysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphs the inhibition of growth of Wein-133 B-cell lymphoma cells after incubation with a monoclonal antibody conjugate of wild-type human carboxypeptidase A, followed by incubation with varying concentrations of the prodrug MTX-phe.

FIG. 2 diagrams the human pancreatic carboxypeptidase A preproenzyme, containing a signal peptide, activation peptide, and mature enzyme.

FIG. 3 depicts the vector pHCPAINS, produced by ligating the HCPA1 NcoI-SalI fragment into the Nco I and Sal I cloning sites of pGEM5zf(-). The HCPA1 Nco I-Sal I fragment encodes amino acids 186-309 of mature HCPA1.

FIG. 4 depicts the vector M13mp19HCPA1, produced by restricting pHCPAINS with Sph I and Sal I to obtain the HCPA1 Nco I-Sal I fragment and an additional 9 base pair pGEM5zf(-) sequence; the Sph I-Sal I fragment was then cloned into M13mp19 using Sph I Sal I cloning sites.

FIG. 5 depicts M13mp19 proHCPA1 mutants containing a 1.2kb fragment of pMP36 HCPA1 (wildtype). Single stranded M13mp19HCPA1 DNA was used for oligonucleotide-directed mutagenesis, to produce mutant forms of HCPA1. A 1.2 kb fragment of pMP36HCPA1 (wildtype) was then cloned into the Nco I site of M13mp19CPA1 mutants.

FIG. 6 depicts pMP36ECPA1 mutant, produced by restricting M13mp19 proHCPA1 mutants with Hind III and Sal I, to obtain a 1.2 kb cDNA fragment encoding the entire proHCPA1 mutant enzyme; this fragment was ligated into the Hind III and Sal I sites of pMP36 to provide pMPHCPA1 mutants that could be further processed for expression in S. cerevisae.

FIG. 7 diagrams pMP vector containing proHCPA cDNA as a fusion with yeast alpha-factor leader, used in expression of HCPA.

DETAILED DESCRIPTION

A mutant human enzyme as used herein shall be taken to be any human enzyme with a sequence differing by at least one amino acid from the amino acid sequence or sequences of that enzyme in the patient to which the therapy is applied.

As used herein the term "cell-type" means any localised or dispersed population of cells possessing a common determinant essentially selective for a pathological state, for example, cancer cells expressing carcinoembryonic antigen, Tag-72, mucin, or the antigens recognised by the antibodies Ing-1, 17-1A, 323/A3, NR-LU-10, c174, PR.1A3, MOV18, G250, U36, E48, NR-CO-02 or any of the other antigens shown in Table 3.

The cell-type specific targetting molecule may be any molecule capable of being noncovalently or covalently linked or conjugated with the mutant enzyme and which can demonstrate a selectivity in its binding affinity for cell-surface markers. Such molecules include polyclonal and monoclonal antibodies, including bispecific antibodies in which one antibody arm noncovalently binds the mutant enzyme and the other performs the cell-type specific targetting (U. Sahin, et al, Cancer Research 50, 6944, 1990).

Alternative targetting molecules which may be linked to mutant mammalian enzymes to form conjugates include hormones, ligands, cytokines, antigens, oligonucleotides and peptidomimetics.

The choice of targetting molecule will depend upon the nature of the cells to be targetted but will most probably be an antibody and preferably a monoclonal antibody as mAbs produce the greatest selectivity.

A "chemotherapeutic agent" which may also be referred to herein as a "drug" includes any molecule which has activity in human therapy. Such chemotherapeutic agents include but are not limited to cytostatic or cytotoxic compounds used in the therapy of cancers or viral infections. A "functionally inactive precursor" which may also be referred to herein as a "prodrug" includes any compound which may be converted into a chemotherapeutic agent under the action of an enzyme. Such functionally inactive precursors may typically be converted to a chemotherapeutic agent by the enzymatic cleavage of the functionally inactive precursor to yield a chemotherapeutic agent and a "prodrug moiety". Such conversion from functionally inactive precursor to chemotherapeutic agent may also occur by enzymatically mediated isomerisation.

A functionally inactive precursor will generally not exhibit clinically significant levels of the therapeutic activity possessed by the chemotherapeutic agent into which it may be enzymatically converted having been chemically derivitized to decrease its normal pharmacological activity. The functionally inactive precursor of a cytotoxic chemotherapeutic agent will not itself exhibit clinically significant cytotoxicity and will be sufficiently stable in vivo such that during therapy, clinically significant level of cytotoxicity are largely only generated at the site of conversion of functionally inactive precursor to chemotherapeutic agent i.e. at the site to which the mutant enzyme has been targetted.

The mutant mammalian enzyme is preferably a wild-type mammalian enzyme with one or more mutations which generate novel substrate specificities without engendering a significant immunological response when administered during human therapy. A significant immunological response may be regarded as one which would preclude the clinical use of such an enzyme in human therapy.

The essential characteristic of a mutant mammalian enzyme of the present invention is the presence of a mutant substrate-binding site irrespective of the amino acid sequences flanking this mutant substrate-binding site. Hence a chimaeric enzyme comprising a mutant substrate-binding site and flanking sequences derived from one or more different enzymes is included with the definitions of a mutant mammalian enzyme of the present invention. Such a chimaeric enzyme may be generated by recombinant DNA technology and/or protein engineering.

Enzymes suitable for directed mutagenesis include any enzymes possessing a catalytic activity capable of converting a prodrug to a drug. Such catalytic activities include transferase, hydrolase, oxido-reductase, isomerase, lyase, or ligase. The directed mutagenesis will generate a novel substrate specificity but preserve the class of catalytic activity involved. For example a mutant isomerase of the present invention will possess a novel isomerase activity sufficiently different from the isomerase from which it was derived to ensure that prodrugs susceptible to activation by the mutant isomerase remain substantially stable in the presence of the isomerase from which the mutant enzyme was derived. As will be shown this dramatic shift in activity may be achieved by the alteration of as little as one residue at the catalytic site. The alteration of the minimum number of residues necessary to obtain the required shift in activity ensures continuity of enzyme structure and hence avoids negative immunological effects when the mutant enzyme is introduced to the body.

A preferred mutant enzyme of the present invention is mutant mammalian carboxypeptidase A (CPA). This enzyme has the general activity of cleaving carboxy-terminal aromatic and aliphatic amino acid residues and has been characterised in a diversity of species and tissue-specific variants. Particularly preferred mutant carboxypeptidases include mutants derived from human pancreatic carboxypeptidase A1 (Catasus L. et al., Biochem. J. 287, 299-303, 1992), human mast cell carboxypeptidase A (Reynolds D. S. et al. J. Clin. Invest. 89 273-282, 1992) and human pancreatic carboxypeptidase A2 (FIG. 9 hereinafter). It will be appreciated that the common characteristic of these carboxypeptidases is the presence of a CPA-like substrate-binding pocket and associated enzymatic activity irrespective of overall sequence or structure of the enzyme and that any enzyme possessing this CPA-like substrate-binding pocket is amenable to mutation to a mutant carboxypeptidase of the present invention.

In a particularly preferred embodiment of the present invention the mutant enzyme is mutant human pancreatic carboxypeptidase A1 or A2 (CPA1 or CPA2) and yet more preferably, CPA1 or CPA2 wherein amino acid substitutions are generated at one or more of residues 203, 210, 242, 244, 250, 253, 255, 267, 268, 269 and 305, of the amino acid sequences shown in Table 4 which represent the wild-type (w.t.) sequences of CPA1 and CPA2 respectively. Particularly preferred combinations of residue substitution include Gly at residues 250 and 268 when 253 and 255 are w.t.; Gly at 253 and 268 when 250 and 255 are w.t.; Gly at 250 and His at 268 when 253 and 255 are w.t.; Gly at 250 when 253, 255 and 268 are w.t.; Ala at 255 and His at 268 when 250 and 253 are w.t. and His at 268 when 250, 253 and 255 are w.t. The most preferred mutants are carboxypeptidase A1 or A2 mutants comprising a single substitution; Gly at 268.

The most preferred conjugate of the present invention comprises all or part of either of mAbs CAMPATH-1H®, 323/A3 or ING-1 and at least the substrate-binding domain of either of mutant mammalian enzymes CPA1 or CPA2 having glycine present at amino acid position 268 as described in Table 5.

Human pancreatic carboxypeptidase A is expressed as a preproenzyme (FIG. 2) which is processed to a proenzyme and subsequently to the mature enzyme. Mutant carboxypeptidases of the present invention may be mutants of the preproenzyme, the proenzyme or of the mature enzyme but are preferably mutants of the mature enzyme. These mutants may be derived from either preproenzyme, the proenzyme or the mature enzyme but are preferably derived from the proenzyme. The mutated proenzyme (or preproenzyme) may then be converted to the corresponding mutant mature enzyme by standard methods such as trypsinisation.

A mutant enzyme of the present invention may be generated from the DNA or RNA source of any enzyme possessing the previously discussed activities by methods well known in the art of molecular biology and more particularly by the methods described hereinafter in the Examples.

The selection of the enzymatic activity and the primary sequence of a mutant enzyme of the present invention will clearly depend upon the nature of the relevant prodrug and, if a prodrug of the type which is activated by the removal of a prodrug moiety, on the precise structure of that moiety.

The substrate-binding or active site of the mutant enzyme must, unlike the corresponding non-mutant enzyme, be capable of interacting with the prodrug in such a way that enzymatic catalysis is facilitated. The ability of the mutant enzyme to perform this activity will depend upon subtle alterations in the 3-dimensional structure of the active site, which is in turn dependent upon the primary amino acid sequence of this region of the protein. Alterations of the primary sequence of an enzyme by standard techniques of protein engineering will allow the generation of an appropriate mutant enzyme for use according to the invention with a corresponding prodrug.

Novel prodrugs comprise those of formula (II) ##STR1## wherein,

X represents NH or O and

R² represents CO₂ H, SO₃ H, SO₂ H, OSO₃ H, PO₃ H₂, OPO₃ H₂, C₁₋₆ alkyl esterified CO₂ H, C₁₋₆ esterified PO₃ H₂, C₄₋₁₂ alkyl, C₄₋₁₂ alkenyl, C₄₋₁₂ alkynyl, C₄₋₁₂ branched alkyl, C₁₋₈ alkyl or C₃₋₈ branched alkyl; substituted with CO₂ H, SO₃ H, SO₂ H, OSO₃ H, PO₃ H₂, OPO₃ H₂, C₁₋₆ alkyl esterified CO₂ H, C₁₋₆ alkyl esterified PO₃ H₂, hydroxyl, alkoxy, halo, haloalkyl, cyano or carboxamide; C₃₋₈ cycloalkyl, aryl or heteroaryl optionally substituted with C₁₋₈ alkyl, C₃₋₈ branched alkyl, C₃₋₈ cycloalkyl, C₃₋₆ alkenyl, C₂₋₆ alkynyl, C₄₋₆ cycloalkenyl, trisubstituted-silyl i.e. (R¹³)(R¹⁴)(R¹⁵)Si, where R¹³, R¹⁴, and R¹⁵ are C₁₋₆ alkyl, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl, C₃₋₆ alkenyl, C₂₋₆ alkynyl, C₄₋₆ cycloalkenyl, aryl, or heteroaryl, and where R¹³, R¹⁴, and R¹⁵ are each the same group or different groups (e.g. trimethylsilyl, tert-butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl), aryl, CO₂ H, SO₃ H, SO₂ H, OSO₃ H, PO₃ H₂, OPO₃ H₂, C₁₋₆ alkyl esterified CO₂ H, C₁₋₆ alkyl esterified PO₃ H₂, carboxamide, hydroxyl, C₁₋₆ alkoxy, C₁₋₆ alkythio, mercapto, halo, haloalkyl, nitro or cyano, R^(2a) and R^(2b) represent H, hydroxy, mercapto, alkoxy, alkylthio, halo, cyano, CO₂ H, SO₃ H, SO₂ H, OSO₃ H, PO₃ H₂, OPO₃ H₂, C₁₋₆ alkyl esterified CO₂ H, carboxamide, C₁₋₆ alkyl esterified PO₃ H₂, cyclic C₂₋₆ [i.e., R^(2a) and R^(2b) together represent (CH₂)₂₋₆ ], trisubstituted-silyl i.e. (R¹³)(R¹⁴)(R¹⁵)Si, where R¹³, R¹⁴, and R¹⁵ are C₁₋₆ alkyl, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl C₃₋₆ alkenyl, C₂₋₆ alkynyl, C₄₋₆ cycloalkenyl, aryl, or heteroaryl and where R¹³, R¹⁴, and R¹⁵ could be the same group or different groups, e.g. trimethylsilyl, tert-butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl; or C₁₋₆ alkyl or C₁₋₆ branched alkyl or C₁₋₆ cycloalkyl or aryl or heteroaryl optionally substituted with hydroxy, mercapto, C_(1-C) alkoxy, C₁₋₆ alkylthio, halo, cyano, CO₂ H, SO₃ H, SO₂ H, OSO₃ H, nitro, PO₃ H₂, OPO₃ H₂, C₁₋₆ alkyl esterified CO₂ H, carboxamide, C₁₋₆ alkyl esterified PO₃ H₂, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ cycloalkyl, trisubstituted-silyl i.e. (R¹³)(R¹⁴)(R¹⁵)Si, where R¹³, R¹⁴, and R¹⁵ are C₁₋₆ alkyl, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl, aryl, or heteroaryl, and where R¹³, R¹⁴, and R¹⁵ could be the same group or different groups, e.g. trimethylsilyl, tertbutyldimethylsilyl or phenyldimethylsilyl; with the proviso that R^(2a) and R^(2b) are not both hydroxy or mercapto.

F comprises a moiety of formula (IV) ##STR2## wherein R¹ represents a group corresponding to the side chain of any α-amino acid for example, H, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl optionally substituted with CO₂ H, C₁₋₆ alkyl esterified CO₂ H, OPO₃, PO₃ H₂, C₁₋₆ alkyl esterified PO₃ H₂, halo, hydroxy, carboxamide, amino optionally substituted with C₁₋₆ alkyl, cyano or nitro,

E represents ##STR3## each of which may be optionally substituted with one or more of hydroxy, one or more of alkoxy, halo, or C₁₋₆ alkyl optionally substituted with one or more of hydroxy, C₁₋₆ alkoxy or halo.

D represents --CH₂ --CH(R³)--, --CH₂ --NR³ --, --NR³ --CH₂ --, --CH₂ S-- or --CH₂ O-- where R³ is H, C₁₋₆ alkyl, allyl or propargyl, optionally substituted with one or more of C₁₋₆ alkoxy halo, hydroxy or cyano; and

B represents ##STR4## wherein,

R⁴ represents H, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl, or C₁₋₆ alkyl;

R⁵ represents C₁₋₆ alkyl, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl, amino (optionally substituted with C₁₋₆ alkyl, C₁₋₆ alkanoyl or benzyl);

R⁶ represents H, C₁₋₆ alkyl, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl, amino (optionally substituted with C₁₋₆ alkyl, C₁₋₆ alkanoyl or benzyl), C₁₋₆ alkoxyl or C₁₋₆ alkylthio; and

R⁷ represents H, C₁₋₆ alkyl, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl, halo C₁₋₆ alkyl or halo

and salts N-oxides, solvates and physiologically functional derivatives thereof; with the proviso that the following compounds are not included within the definition of novel prodrugs although the use of these compounds in therapy does form one embodiment of the present invention;

N-(N-(4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoyl)-L-glutam-1-yl)-L-glutamic acid,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamic acid,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-aspartic acid,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-phenylalanine,

4-bis(2-chloroethyl)amino)-phenylalanylphenylalanine and

4-bis(2-chloroethyl)amino)-phenylalanyl-3,5-dimethyl-4-methoxyphenylalanine

In a preferred embodiment of prodrugs according to the present invention,

X is NH;

R² is C₃₋₈ cycloalkyl or aryl substituted with C₁₋₈ alkyl, C₃₋₈ cycloalkyl, C₃₋₉ branched alkyl or trisubstituted-silyl i.e. (R¹³)(R¹⁴)(R¹⁵)Si, where R¹³, R¹⁴, and R¹⁵ are C₁₋₆ alkyl, C₃₋₆ branched alkyl, C₃₋₆ cycloalkyl, aryl, or heteroaryl, and where R¹³, R¹⁴, and R¹⁵ are each the same group or different groups (e.g. trimethylsilyl, tertbutyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl);

R^(2a) and R^(2b) are both H;

R¹ is H, C₁₋₆ alkyl, CH₂ CO₂ H, CH₂ CH₂ CO₂ H or CH₂,CH₂ CH₂ NH₂,

E is ##STR5##

D is --CH₂ NH--, --CH₂ N(CH₃)--, --CH₂ N(CH₂ C.tbd.CH)--, --CH₂ CH₂ -- or --CH₂ --CH(C₂ H₅)--

and B is ##STR6## and salts, N-oxides, solvates and physiologically functional derivatives thereof.

In the most preferred embodiment of prodrugs according to the present invention,

X is NH;

R² is phenyl or para-hydroxyphenyl substituted with cyclopentyl, cyclobutyl or tert-butyl. The cyclopentyl, cyclobutyl and tert-butyl groups may both be either ortho- or meta- but are preferably meta-, R^(2a) and R^(2b) are H,

R¹ is CH₂ CH₂ CO₂ H

E is ##STR7##

and B is ##STR8## and salts, N-oxides, solvates and physiologically functional derivatives thereof.

Where chiral centres of prodrugs as defined above are present they may, independently, each be either in the S or R configuration or may be a mixture of the S and R configuration. Preferably they are in the S-configuration for `F` and the chiral carbon of formula (II) adjacent to X.

A preferable prodrug for use according to the present invention is a prodrug of the cytotoxic agent melphalan (UK 750, 155) which is commercially available under the name Alkeran (T. M. The Wellcome Foundation Limited) and has the chemical name 4-[bis(2-chloroethyl)amino]-1-phenylalanine. Prodrugs of melphalan according to the present invention have the general formula ##STR9## wherein R², R^(2a), R^(2b) and X are as herein defined.

The following are particularly preferred novel prodrugs according to the present invention:

N-((S)-4-carboxy-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(F)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)butanoyl)-L-phenylalanine,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-phenylalanine,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclobutyl-L-phenylalanine,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-trimethylsilyl-L-phenylalanine,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-phenylalanine,

N-(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine,

N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(F)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine,

N-(4-(((2,4-diamino-6-pteridinyl-methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-tyrosine,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3,5-diiodo-L-tyrosine,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-tyrosine,

(S)-3-(3-cyclopentyl-4-hydroxyphenyl)-2-((N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)oxy)propionic acid,

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-tyrosine

and salts, N-oxides, solvates and physiologically functional derivatives thereof.

The most preferred prodrug according to the present invention is:

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-tyrosine.

Any chiral centers of any of the chemical structures on compounds described herein can be in the R and S configuration. Although these chiral centers of the chemical structures drawn hereinafter which are depicted in the preferred S-configuration, it is understood that the corresponding chemical structures having the chiral centers in the R-configuration as well as enantiomeric and diastereomeric mixtures of these compounds are also considered as being party of the invention.

A prodrug of formula (V), where B, D and E are as defined in formula (III), and X, R¹ and R² are as defined in formulas (IV) and (II), ##STR10## can be prepared by hydrolysis of an ester of formula (VI); where R⁸ is alkyl, branched alkyl, or cycloalkyl group such as methyl, ethyl, tert-butyl or cyclohexyl; or an aryl group such as phenyl, 2,6-dimethylphenyl; benzyl (optionally substituted with halo, alkoxy, or alkyl groups such as methyl or tert-butyl); or 9-fluorenylmethyl (Fm); normally in the presence of a base, such as NaOH, LiOH; or, in the case where R⁸ is Fm, diethylamine or piperidine; or in the presence of an acid such as HCl or trifluoroacetic acid; in a solvent or solvent mixture such as THF, nitromethane or THF:H₂ O; at a temperature, for example 0° C. to reflux temperature, conveniently at room temperature; or in the present of an acid, such as HCl or trifluoroacetic acid; ##STR11## in a solvent such as THF or nitromethane; at a temperature, for example 0° C. to reflux temperature, conveniently at room temperature; or by hydrogenolysis where R⁸ is 9-fluorenylmethyl (Fm) or benzyl optionally substituted with alkyl groups such as methyl or tert-butyl using hydrogen at ambient or elevated pressure, for example between 1·75 kgcm⁻² and 7·0 kgcm⁻², conveniently at 3·5 kgcm⁻², in the presence of a catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol, or a mixture of methanol and dichloromethane, at ambient temperature, or by methods which are known to anyone skilled in the art such as those methods described in Rylander, P. N., Catalytic Hydrogenation in Organic Syntheses, Academic Press: New York 1979, or in Rylander, P. N., Hydrogenation Methods, Academic Press: London, 1985, or in Bodanszky, M., et al., The Practice of Peptide Synthesis, Springer-Verlag: Berlin, 1984, which are incorporated herein by reference; or by oxidation where R⁸ is phenyl or benzyl substituted with alkoxy (preferably in the 2-, 4-, or 6- position of the phenyl ring) optionally substituted with suitable alkyl groups such as methyl or tert-butyl, in the presence of a suitable oxidizing agent such as ceric ammonium nitrate or dichlorodicyanoquinone (DDQ) in a suitable solvent such as methanol, water, dichloromethane, or combinations thereof, by methods which are known to anyone skilled in the art such as those methods described in Heathcock, C. H., et.al., Tetrahedron, 1981, 37, 4087; or those methods described in Greene, T. W., et.al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference; or where R⁸ is 2-(trimethylsilyl)ethyl by reaction with a source of a suitable nucleophile such as potassium fluoride or tetrabutylammonium fluoride for which methods are known to anyone skilled in the art such as those methods described in Greene, T. W., et al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, or in Bodanszky, M., et.al., the Practice of Peptide Synthesis, Springer-Verlag: Berlin, 1984, or in Jones, J., The Chemical Synthesis of Peptides, Clarendon Press: Oxford, England, 1991, which are incorporated herein by reference; or where R⁸ is a photoremovable group such as orthonitrophenyl by photolytic methods which are known to anyone skilled in the art such as those described in Cama, L. D., et.al., J. Am. Chem. Soc., 1978, 100, 8006, or in Pillai, V. N. R., Synthesis, 1980, 1, or in Pillai, V. N. R., Org. Photochem., 1987, 9, 225 which are incorporated herein by reference. Esters present on sidechains R¹ or R² may or may not be hydrolyzed in the same reaction utilised to hydrolyze R⁸, depending on the choice of ester group and the reaction conditions.

Ester (VI) can be prepared by reaction of an amine of formula (VII) where R¹ is defined as in formula (IV), R² and X are defined as in formula (II), and R⁸ is defined as in formula (VI); a carboxylic acid of formula (VIII), ##STR12## where B and D are as defined in hereinbefore and G represents a group (IX), ##STR13## where n=3-7, or 1,4-cyclohexyl(X) where the 1- and 4-substituents may either in the cis- or trans- configuration or mixtures of the two configurations, or an aromatic group chosen from the following: ##STR14##

The reaction of amines of formula (VII) with carboxylic acids of formula (VIII) are carried out in the presence of a coupling agent; including but not limited to diethyl cyanophosphate (XI), 1-ethyl-3-(dimethylaminopropyl)carbodiimide (XII), or dicyclohexylcarbodiimide (XIIa), optionally in the presence of an activating group such as 1-hydroxybenzotriazole (HOBT), in a polar, aprotic solvent, conveniently N,N-dimethylformamide (DMF) or N,N-dimethylpropyleneurea (DMPU) at a temperature, for example 0° C. or 80° C., ##STR15## conveniently at room temperature. Alternatively, an ester of formula (VI) can be prepared by treating a mixture of carboxylic acid (VIII) and a tertiary amine base; such as triethylamine, diisopropylethylamine, or N-methylmorpholine; in an aprotic solvent, such as DMF or DMPU; at a temperature from 0° C. to 25° C.; with a acylating agent, such as isobutyl chloroformate or ethyl chloroformate; followed by addition of amine (VII).

Several of the preferred carboxylic acids (VIII) can be purchased from commercial suppliers, eg. (XIII) can be purchased from Aldrich Chemical Co; or can be prepared via literature methods, eg. (XIV): De Graw, J I, et al, J. Med Chem, 1982, 25, 1227; Ibid, 1986, 29, 1056. (XV): Jones, T R, et al,: J. Med Chem, 1986, 29, 1114; Nair, M G, et al,: Ibid, 1986, 29, 1754; Ghazola, M, et al,: Ibid, 1986, 29, 1263; Acharya S. P. et.al., J. Heterocyclic Chem, 1975, 12, 1283. (XVI): Barnett, C J, et al, Tetrahedron Lett, 1989, 30, 6291. (XVII): Styles V L, et al,: J. Med Chem, 1990, 33, 561 Taylor, E.c., et al. J. Med. Chem. 1992, 32 1517: (XVIIa) and Bisset, G. M. F. et al J. Med. Chem, 1992 35 859; ##STR16##

Carboxylic acids of formula (VIII) can be prepared by hydrolysis of esters of formula (XVIII), wherein B and D are as defined in formula (III), G is as defined in formula (VIII) and R⁹ is a suitable group such as methyl, ethyl, or tert-butyl; normally in the presence of a suitable base, such as NaOH or LiOH; or a suitable acid, such as HCl or trifluoroacetic acid; in a suitable solvent or solvent mixture, such as THF, nitromethane, or THF:H₂ O; at a temperature, for example from 0° C. to reflux temperature, conveniently at room temperature.

    B--D--G--CO.sub.2 --R.sup.9                                (XVIII)

For prodrugs where D in formula (III) or formula (XVIII) represents --CH₂ --NR³ --, esters of formula (XVIII) can be prepared by:

(a) reaction of a compound of formula (XIX), where G is as defined in formula (VIII), R⁹ is as defined in formula (XVIII), and R¹⁰ represents

    R.sup.10 --G--CO.sub.2 R.sup.9                             (XIX)

NHR³ wherein R³ is as defined in formula (III); and a compound of formula (XX)

    K--R.sup.11                                                (XX)

wherein R¹¹ is an aldehyde group or cyano group and K is a heterocyclic group such as those defined as B in formula (III) or a group such as (XXI) where Z═O, or a group such as defined as B in formula (III) or a group such as (XXI) where Z═O substituted with a suitable protecting group to provide a more suitable reactant, suitable groups including but not limited to N-pivaloyl, N-benzoyl, N-carbobenzyloxy (N-cbz), or N-tert-butoxycarbonyl (N-T-BOC). Such protecting groups can be removed at a later stage in the synthesis, where appropriate, using standard methods known to anyone skilled in the art such as those methods described in Green, T. W., et al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference. ##STR17## Esters of formula (XVIII) are prepared by reaction of (XVI) and (XX) in a solvent such as THF, DMPU, DMF, ethanol, or acetic acid at a temperature between 25° C. and 100° C., optionally with removal of water during the course of the reaction, followed by reduction with sodium cyanoborohydride or hydrogen gas in the presence of a suitable catalyst, such as Raney nickel, using standard reaction conditions known to anyone skilled in the art.

(b) reaction of a compound of formula (XIX), as defined in (a), with a compound of formula (XX), where K is as defined in (a) but R¹¹ is defined as CH₂ Y, where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl, in a suitable solvent such as THF, in the presence of a base such as NaHCO₃, Na₂ CO₃, K₂ CO₃, triethylamine, or diisopropylethylamine, at an elevated temperature, for example 50° C. to 150° C. and conveniently 80° C. to 120° C.

For prodrugs where D in formula (III) and formula (XVIII) represents --NR³ --CH₂ --, esters of formula (XVIII) can be prepared by reaction of a compound of formula (XIX) where R¹⁰ represents an aldehyde group and G is as defined in formula (VIII), and a compound of formula (XX), where K is as defined in formula (XX) and R¹¹ is defined as HNR³ where R³ is as defined in formula (III), in a solvent such as THF, DMF, DMPU, or acetic acid at a temperature between 25° C. and 100° C., optionally with removal of water during the course of the reaction, followed by reduction with sodium cyanoborohydride or hydrogen gas in the presence of a suitable catalyst, such as Raney nickel.

For prodrugs where D in formula (III) and formula (XVIII) represents --CH₂ O-- or --CH₂ S-- esters of formula (XVIII) are prepared by reaction of a compound of formula (XIX), where R⁹ is as defined in formula (XVIII), G is as defined in formula (VIII), and R¹⁰ is OH or SH; with a compound of formula (XX), where K is as defined in formula (XX) and R¹¹ is CH₂ Y, where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; in a suitable solvent such as DMF, DMPU, or acetone; in the presence of a base such as triethylamine, diisopropylethylamine, cesium carbonate, cesium bicarbonate, sodium carbonate, or sodium bicarbonate, at a temperature from 25° C. to 80° C.

For prodrugs where D in formula (III) and formula (XVIII) represents --CH₂ CH₂ --, esters of formula (XVIII) are prepared by:

(a) reaction of a compound of formula (XX) where K is defined as in formula (XX) and where R¹¹ is CH₂ Br, with triphenylphosphine in a polar solvent, for example a C₁₋₄ alkanol or glycol, conveniently methanol or ethanol, normally in the presence of a base, for example a metal alkoxide conveniently formed from the metal and the solvent, ie. sodium methoxide or sodium ethoxide at a temperature of 0° C. to reflux temperature; followed by addition of a compound of formula (XIX), where R⁹ is as defined in formula (XVIII), G is as defined in formula (VIII), and R¹⁰ is an aldehyde group; followed by reduction with hydrogen at elevated pressure, for example between 1·75 kgcm⁻² and 7·0 kgcm⁻², conveniently at 3·5 kgcm² in the presence of a suitable catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol or a mixture of methanol and dichloromethane, at ambient temperature.

(b) reaction of a compound of formula (XIX) where R⁹ is defined as in formula (XVIII) and G is defined as in formula (VIII), and R¹⁰ is CH₂ Br, with triphenylphosphine in a polar solvent, for example a C₁₋₄ alkanol or glycol, conveniently methanol or ethanol, normally in the presence of a base, for example a metal alkoxide conveniently formed from the metal and the solvent, ie. sodium methoxide or sodium ethoxide at a temperature of 0° C. to reflux temperature; followed by addition of a compound of formula (XX), where K is as defined in formula (XX), and R¹¹ is an aldehyde group; followed by reduction with hydrogen at elevated pressure, for example between 1·75 kgcm⁻² and 7·0 kgcm⁻², conveniently at 3·5 kgcm⁻², in the presence of a suitable catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol or a mixture of methanol and dichloromethane, at ambient temperature.

(c) reaction of a compound of formula (XX), where K is defined as in formula (XX) and R¹¹ is chloro, bromo, or iodo; with a compound of formula (XIX), where R⁹ is defined as in formula (XVIII), G is as defined in formula (VIII), and R¹⁰ is a acetylene group C.tbd.CH.

The reaction is carried out in the presence of a Pd(O) catalyst, conveniently tetrakis-(triphenylphosphine)palladium; and a base such as triethylamine or diisopropylethyl-amine; in a polar aprotic solvent such as DMF or DMPU; at a temperature of 25° C. to 50° C.; followed by reduction with hydrogen at elevated pressure, for example between 1.75 kgcm⁻² and 7.0 kgcm⁻², conveniently at 3.5 kgcm⁻², in the presence of a suitable catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol or a mixture of methanol and dichloromethane, at ambient temperature.

For prodrugs where D in formula (III) and formula (XVIII) represents --CH₂ --CH(R³)--, R³ is as defined in formula (III), esters of formula (XVIII) are prepared by reaction of a compound of formula (XX), where K is defined as in formula (XX) and where R¹¹ is CH₂ Br, with tributylphosphine or triphenylphosphine in a polar solvent, for example a C₁₋₄ alkanol or glycol, conveniently methanol or ethanol, normally in the presence of a base, for example a metal alkoxide conveniently formed from the metal and the solvent, ie. sodium methoxide or sodium ethoxide, or in a solution of dimethyl sulfoxide and a base such as sodium hydride at a temperature from 25° C. to 80° C.; followed by addition of a compound of formula (XI), where R⁹ is defined as in formula (XVIII), G is defined as in formula (VIII), and R¹⁰ is a keto group --(CO)--R³ where R³ is defined as in formula (III); subsequently stirring the reaction mixture at a temperature from 25° C. to reflux temperature for an extended period from 4 to 96 hours, conveniently 24 to 48 hours; followed by reduction with hydrogen at elevated pressure, for example between 1.75 kgcm⁻² and 7.0 kgcm², conveniently at 3.5 kgcm⁻², in the presence of a suitable catalyst such as palladium-on-carbon or platinum oxide, in a polar solvent, for example methanol ethanol or acetic acid.

Compounds of formula (XX) where K is as defined above and R¹¹ is CH₂ Br can be prepared using methodology available to those skilled in the art. (Piper, J R et al, J Org Chem, 1977, 42, 208; Piper, J R et at, J Med Chem, 1986, 29, 1080; Oakes V, et al J Chem Soc, 1956, 4433Acharya, S P, et al, J Heterocyclis Chem, 1975 12, 1283; bird, O D et al in Pteridine Chemistry, Pfleiderer W & Taylor E C, Eds, Pergamon Press, Oxford, 1964, p. 417; Piper, J R, et al J Heterocyclic Chem, 1974, 279).

Compounds of formula (XX) where K is as defined above and R¹¹ is an aldehyde group can be prepared using methodology available to those skilled in the art. (Taylor, E C, et al, J Org Chem, 1983, 48, 4852; Arnold. A, et al, Collect Czech Chem Commun, 1960, 25, 1318; Beardsley, G P, et al Proc Am Assoc Cancer Res, 1986, 1027.)

Compounds of formula (XX) where K is as defined above and R¹¹ is a cyano group can be prepared using methodology available to those skilled in the art, (Elsager, E F, et al, Lect Heterocyclic Chem, 1974, 2, S-97; Davoll, J, et al, J Chem Soc (C), 1970, 997; Jones, T, R., Eur, J, Cancer, 1981, 17, 11; Kondo, T., et,al., Chem Lett., 1980, 559.

Compounds of formula (XX) where K is as defined above and R¹¹ is an amino group can be prepared using methodolgy available to those skilled in the art, (Hynes, J B, et al, J Med Chem, 1975, 18, 632, 1191; Davoll, J, et al, J Med Chem, 1972, 15, 837; Bernetti, R et al, J Org Chem, 1962, 27, 2863.)

Compounds of formula (XX) where K is as defined above and R¹¹ is a bromo, chloro, or iodo group can be prepared using methodology available to those skilled in the art, Taylor, E C, et al, J Med Chem, 1992, 35, 4450; Taylor, E C. et al, J Org Chem, 1989, 54, 3618; Jones, J H, et al, J Med Chem, 1968, 11, 322.)

Carboxylic acids of formula (VIII) where A is a group of formula (XXI) where Z═S, can be prepared from carboxylic acids of formula (VIII) where C is a group of formula (XXI) wherein Z═O, by reaction with a sulfurating agent such as P₂ S₅ in a solvent such as pyridine at a temperature between 25° C. and reflux temperature.

Compounds of formula (XIX) where G is defined as in formula (VIII), R⁹ is as defined in formula (XVIII), and R¹⁰ is NHR ³ is as defined in formula (III) can be prepared from compounds of formula (XIX) where G and R⁹ are as defined above and R¹⁰ is NH₂ by reaction with a compound R³ --Y where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; in the presence of a suitable base such as 2,6-lutidine, in a polar aprotic solvent such as DMF or dimethylacetamide at an elevated temperature between 50° C. and 100° C.

Compounds of formula (XIX) where G is a phenyl or furyl group, R¹⁰ is NH₂, and R⁹ is defined as in formula (XVIII) can be obtained from commercial suppliers, or can be readily prepared from such compounds that are available from commercial suppliers using methods known to anyone skilled in the art. One such preferred compound of formula (XXII) ##STR18## can be prepared by methods described by N Soundararajan and M S Platz. (soundararajan N et al, J Org Chem, 1990, 55, 2034.) One such preferred compound is of formula (XXIIa) and can be prepared by methods described in Mackay, D., Can J. Chem, 1966, 44, 2881: or in Paul, H., et al., Arch, Pharm, 1978, 52 538. ##STR19##

Compounds of formula (XIX) where G is a cyclohexyl group, R¹⁰ is NH₂, and R⁹ is as defined in formula (XVIII) can be obtained from commercial suppliers, or can be readily prepared using standard methods available to anyone skilled in the art, (Banfi, A, et al, Synth Commun, 198, 19, 1787; Krieg, R, et al, J Prakt Chem, 1987, 329, 1123; Johnston, T P, et al, J Med Chem, 1977, 20, 279.)

Compounds of formula (XIX) where G is a thienyl group, R¹⁰ is NH₂, and R⁹ is as defined in formula (XVIII) can be prepared by methodolgy available to anyone skilled in the art. (Goddard, C J, J Heterocyclic Chem, 1991, 28, 17Decroix, B, et al, J Chem Res (S), 1978, 134; Paul, H, et al, Arch Pharm, 1978, 311, 679; Mackay, D, Can J Chem, 1966, 44, 2881.)

Compounds of formula (XIX) where G is a thiazole group, R¹⁰ is NH₂, and R⁹ is as defined in formula (XVIII) can be prepared from 5-nitrothiazole-2-carboxylic acid (Strehlke, P, Chem Ber, 1973, 106, 721) using esterification and reduction methods known to anyone skilled in the art.

Compounds of formula (XIX) where G is a pyridyl group, R¹⁰ is NH₂, and R⁹ is as defined in formula (XVIII) can be prepared using methodology available to anyone skilled in the art. (Dawson, M I, et al, J Med Chem, 1983, 26, 1282; Finch, N et al, J Med Chem, 1978, 21, 1269; Cooper, G H, et al, J Chem Soc(C), 1971, 3257; Deady, L W, et al, Aust J Chem, 1971, 24, 385.)

Compounds of formula (XIX) where G is a pyrimidyl group, R¹⁰ is NH₂, and R⁹ is as defined in formula (XVIII) can be prepared using methods analogous to those described by P R Marsham, et al. (Marsham, P R, et al, J Med Chem, 1991, 34, 1594.)

General methods for synthesis of compounds of formula (VIII) are described in two recent reviews, (Palmer, D C, et al, Prog Med Chem, 1988, 25, 85; Rosowsky, A, Prog Med Chem, 1989, 26, 1.)

Esters of formula (VI) where E is defined as ##STR20## can be prepared by reaction of a compound of formula (XX) where R¹¹ is CH₂ Br; and a compound of formula (XXIII) where R¹ is defined ##STR21## as in formula (IV), R² and X are defined as in formula (II), and R⁸ is defined as in formula (VI); in a polar solvent, such as DMF or DMPU; in the presence of a base, such as sodium bicarbonate, cesium bicarbonate, sodium carbonate or cesium carbonate; at an elevated temperature, for example form 50° C. to 100° C., conveniently at 100° C. Compounds of formula (XXIII) can be prepared from compounds of formula (VII) and a compound of formula (XXIV) by methods analgous to those described by ##STR22##

W Pendergast, et al, (Pendergast, W, et al, J Med Chem, [1994], 37 p 838) or in patent application WO 01/19700 which is incorporated in its entirely by reference herein.

Compounds of formula (VII) can be prepared from compounds of formula (XXV), where R¹ is as defined in formula (IV), R² and X are as defined in formula (II), R⁸ is as defined in formula (VI), and R¹² represents a suitable protecting group such as N-tert-butoxycarbonyl(N-t-Boc), or N-carbobenzyloxy (n-Cbz), for which methods are known to anyone skilled in the art such as those methods described in Greene, T. W., et al., Protective Groups in Organis Synthesis, second edition, John Wiley & Sons; New York, 1991, which are incorporated herein by reference.

Compounds of formula (XXV) ##STR23## can be prepared by a reaction of carboxylic acid of formula (XXVI) and a compound of formula (XXVII), where X is defined as in formula (II); for compounds where X is NH, compounds of formula (XXV) can be prepared by reaction for a carboxylic acid of formula (XXVI) and an amine of formula (XXVII), where X is NH, in the presence of a suitable coupling agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or dicyclohexylcarbodiimide, in the presence of a suitable base such as N-methylmorpholine, in an aprotic solvent such as dichloromethane or DMF at 0° C. to 50° C. or by methods which are known to anyone skilled in the art such as those methods described in Bodanszky, M., et,al., The Practice of Peptide Synthesis, Springer-Verlag: Berlin, 1984, or in Jones, J., The Chemical Synthesis of Peptides, Clarendon Press: Oxford, England, 1991which are incorporated herein by reference. ##STR24## dichloromethane or DMF at 0° C. to 50° C. for which methods are known to anyone skilled in the art such as those methods described in Green, T. W., et.al., Protective Groups in Organic Synthesis, second edition John Wiley & Sons: New York 1991, which are incorporated herein by reference.

For compounds of formula (XXV) where X is O, compounds of formula (XXV) can be prepared by reaction of a carboxylic acid of formula (XXVI) with a suitable activating agent such as para-toluenesulfonyl chloride or benzenesulfonyl chloride, in a suitable solvent such as pyridine or lutidine, at a temperature between 0° C. and 25° C.; followed by addition of an alcohol of formula (XXVII) where X is O. ##STR25##

Esters of formula (XXVII), where X is defined as in formula (II), can be prepared from carboxylic acids of formula (XXVIII) using standard methods known to anyone skilled in the art for which methods are known to anyone skilled in the art such as those methods described in Green, T. W., et.al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference.

Carboxylic acids of formula (XXVIII), where X is O, can be made from carboxylic acids of formula (XXVIII) where X is NH by methods analogous to those described by C Morin, et al, (Morin, C, et al, Synthesis, 1987, 479) and K Mori, et al (Mori, K, et al, Tehrahedron, 1982, 38, 3705).

Carboxylic acids of formula (XXVIII) where X is NH can be prepared from compounds of formula (XXIX); where W is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl and R² is defined as in formula (II); by methods analogous to

    WCH.sub.2 R.sup.2                                          (XXIX)

described by R M Williams (Williams, R M, Aldrichimica Acta, 1992, 25, 11 and references cited therein), or by M J O'Donnell, et al, (O'Donnell, M J, et al, J Am Chem Soc, 1989, 111, 2353.)

Compounds of formula (XXIX), where W is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl can be prepared from compounds of formula (XXIX) where W is a hydroxyl group for which methods are known to anyone skilled in the art such a those methods described in March, J., Advanced Organic Chemistry, fourth edition, John Wiley & Sons: New York, 1992, which are incorporated herein by reference.

Some of the preferred compounds of formula (XXIX) where R² is a phenyl group substituted with C₁₋₈ alkyl, C₃₋₈ branched alkyl, or C₃₋₈ cycloalkyl can be prepared by reaction of a compound of (XXIX) where W is a hydroxyl group, or a hydroxyl group with a suitable protecting group which can be attached and later removed where appropriate in the synthesis, by standard methods known to anyone skilled in the art, and R² is a phenyl group substituted with an iodo group or a bromo group; the reaction can proceed by addition of a suitable olefinic compound in the presence of a PD catalyst by methods analogous to those described by H A Dieck, et al, (Dieck, H A, et al, J Am Chem Soc, 1974, 96, 1133), followed by reduction with hydrogen gas in the presence of a suitable catalyst using standard condition known to anyone skilled on the art for which methods are known to anyone skilled in the art such as those methods described in Rylander, P. N., Catalytic Hydrogenation in Organic Syntheses, Academic Press: London, 1985, which are incorporated herein by reference; or the reaction can proceed by reaction with a suitable aklyllithium reagent such as n-butyllithium reagent, in an ethereal solvent such as THF, at a reduced temperature, for example -100° C. to -20° C., conveniently at -78° C., followed by addition of the resulting benzylonic alcohol group by methods analogous to those described by C T West, et al, (West C T, et al, J Org Chem, 1973, 38, 2675).

Some of the preferred compounds of formula (XXIX) where R² is a phenyl group substituted with a carboxyl or esterified carboxyl group can be prepared by reaction of a compound of formula (XXIX) where W is a hydroxyl group and R² is a phenyl group substituted with a bromo or iodo group, with carbon monoxide in a suitable alkanol solvent by methods analogous to those described by A Schoenber, et al, (Schoenber, A, et al, J Org Chem, 197, 39, 3318), followed by hydrolysis of the resulting ester, if desired, for which methods are known to anyone skilled in the art such as those methods described in Greene, T, W., et,al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference.

Some of the preferred carboxylic acids of formula (XXVIII) where X is NH and R² is a para-hydroxphenyl) group substituted with C₁₋₈ alkyl, C₃₋₈ branched alkyl, or C₃₋₈ cycloalkyl can be prepared by reaction to tyrosine substituted on the phenyl ring with a bromo or an iodo group; where the amino and/or carboxyl groups can be optionally substituted with a suitable protecting group which can be attached and later removed where appropriated in the synthesis, for which methods are known to anyone skilled in the art such as those methods described in Greene, T. W., et.al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York 1991which are incorporated herein by reference with a suitable olefinic compound in the presence of a Pd catalyst by methods analogous to those described by H A Dieck, et al, (Dieck, H A, et al, J Am Chem Soc, 1974, 96, 1133), followed by reduction with hydrogen gas in the presence of a suitable catalyst using standard conditions known to anyone skilled in the art.

Some of the preferred carboxylic acids of formula (XXVIII) where X is NH and R² is a para-hydroxyphenyl) group substituted with carboxyl or esterified carboxyl can be prepared by reaction to tyrosine substituted on the phenyl ring with a bromo or an iodo group; where the amino and/or carboxyl groups can be optionally substituted with a suitable protecting group which can be attached and later removed where appropriate in the synthesis, for which methods are known to anyone skilled in the art such as those methods described in Green, T. W., et. al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference; with carbon monoxide in a suitable alkanol solvent by methods analogous to those described by (Schoenberg, A, et al, J Org Chem, 1974, 39, 3318), followed by hydrolysis of the resulting ester, if desired, by using standard methods known to anyone skilled in the art.

An alternate method for the synthesis of compounds of formula (VI) involves reaction of a compound of formula (XX), where K is as defined in formula (XX) and R¹¹ is an aldehyde group, a cyano group, NHR³ where R³ is as defined in formula (III), or a group CH₂ Y where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; with a compound of formula (XXX), where R¹ is as defined in formula (IV), G is as defined in formula (VIII), and R¹⁰ is defined as Oh, SH, NHR³ where R³ is as defined in formula (III), or a group CH₂ Y where Y is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; by methods analogous to ##STR26## those described for the preparation of compounds of formula (XVIII) by reaction of compounds of formula (XX) with compounds of formula (XIX).

Compounds of formula (XXX) where R¹ is as defined in formula (IV), R² and X are as defined in formula (II), R⁸ is as defined in formula (VI), G is defined as in formula (VIII), and R¹⁰ is CH₂ Y where Y is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl can be prepared from compounds of formula (XXX) where R¹, R², R⁸, X and G are as defined above, and Where R¹⁰ is CH₂ OH for which methods are known to anyone skilled in the art such as those methods described in March, J., Advanced Organic Chemistry, fourth edition, John Wiley & sons: New York, 1992which are incorporated herein by reference.

Compounds of formula (XXX), where R¹ is as defined in formula (IV), R² and X are as defined in formula (II), R⁸ is as defined in formula (VI), G is as defined in formula (VIII), and R¹⁰ is defined as OH, SH, CH₂ OH, NHR³ where R³ is as defined in formula (III), an aldehyde group, or a keto group --(CO)--R³ can be prepared from compounds of formula (VII), where R¹, R², R⁸, and X are as defined above, with a compound of formula (XIX), where R⁹ is hydrogen and R¹⁰ is as defined above, by methods analogous to those described from the preparation of compounds of formula (VI) by reaction of compounds of formula (VII) with compounds of formula (VIII), In cases where R¹⁰ is OH, SH, Ch₂ OH, NHr³, it may be preferable to add a protecting group to R¹⁰ prior to reaction with a compound of formula (VII), Such protecting groups can be added and can be removed at a later stage in the synthesis, for which methods are known to anyone skilled in the art such as those methods described in Green, T. W., et,al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference.

Compounds of formula (XIX), where R⁹ is hydrogen and R¹⁰ is defined as OH, SH, CH₂ OH, NHR³ is as defined in formula (III), an aldehyde group, or a keto group --(CO)--R³ above, can be prepared from compounds of formula (XIX), where R⁹ is as defined in formula (XVIII) and R¹⁰ is as defined above, for which methods are known to anyone skilled in the art such as those methods described in Green, T. W., et.al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference.

Prodrugs of general structure (XXXI) where X and R² are as defined in formula (II) can be prepared from compounds of formula (XXXII) ##STR27## wherein R¹² is as defined in formula (XXV), R² is as defined in formula (II), and R⁸ is as defined in formula (VI), for which methods are known to anyone skilled in the art such as those methods described in Greene, T. W., et.al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference. ##STR28##

Compounds of formula (XXXII) can be prepared by reaction of compounds of formula (XXXIII) where R¹² is as defined in formula (XXV); ##STR29## with compounds of formula (XXVII) where X is NH or O, R² is as defined in formula (II), and R⁸ is as defined in formula (VI); using methods analogous to those described for the preparation of compounds of formula (XXV) from compounds of formulas (XXVI) and (XXVII).

Compounds of formula (XXXIII), where R¹² is a suitable protecting group as defined in formula (XXV), can be prepared for which methods are known to anyone skilled in the art such as those methods described in Green, T. W., et.al., Protective Groups in Organic Synthesis, second edition, John Wiley & Sons: New York, 1991, which are incorporated herein by reference; from melphalan (UK 750, 155) which is commercially available under the name Alkeran (T.M. The Wellcome Foundation Limited) and has the chemical name of 4-[bis(2-chloroethyl)amino]-1- phenylalanine.

The term protein engineering has come to mean the structurally directed modification of a protein to obtain a specific, desired new set of protein properties or, in this context, a precise enzymatic activity, Two approaches t modification of protein specificity and reactivity can be used. The differences in these approaches depend upon the chemical and structural information available for a specific enzyme. If no chemical structural information is known about a protein, random mutations can be introduced singly or in groups to determine the importance of specific amino acid residues using techniques well known in the art of molecular biology. However, a more directed approach using site directed mutagenesis, can be performed, if there is detailed chemical and structural information available on a protein. This involves only specific changes to amino acid residues that the chemical and structural data suggest are the residues critical to determination of the desired enzymatic activity, If 3-dimensional structural data are available, the methodolgy can be carried further to provide molecular models of proposed mutation. These models can provide estimates of the relative stability or reactivity of a specific site directed mutation.

Typically, in the computer-assisted protein engineering approach, computer models based upon x-ray crystallographic structures or models built, by homology model building, from x-ray structures of homologous proteins are used. In many cases the starting crystallographic structures can be obtained from the publicly available Brookhaven Protein Database.

Extensive studies of protein structure and the function of individual amino acids have allowed the classification of residues in respect of their importance in structure, stability, reactivity or catalysis. Many active site amino acid residues in proteins show unusual chemical reactivity, these chemical reactivities characterise the catalytic properties of proteins. This chemical reactivity aids in the identification of the active site and the non-reactive associated residues in the active site. Crystallographic studies in concert with chemical reactivity information can indicate distinctly the active site locations. Additionally, some proteins will crystallise with substrates or inhibitors in the active site. All of these pieces of structural information can be combined to enhance the focus of design efforts towards specific residues as candidates for site directed mutagenesis.

The specific desired change in protein reactivity can be obtained by specific changes in the amino acid at a given site of interest. Amino acid residues can be inserted, thus separating functional groups in space, or existing residues can be deleted, bringing functional groups closer together.

Additionally, changes in protein structure can be effected by changing the side chain or main chain torsion angles and the functional groups on the amino acid residues. These torsion angles can be changed by exchange, deletion or insertion of amino acid residues. If structural data are available, all these changes can be modelled using computer-assisted molecular modelling techniques. Forces interacting on the protein structures can be modelled in the computer using molecular mechanics or molecular dynamics force fields.

The space available for ligand binding within a protein can be modified by changes in the bulk of amino acid side chains. Mutating bulky residues, such as isoleucine or leucine, to alanin or glycine, can provide pockets within a ligand binding site without appreciable changing the hydrophobicity of the site. The opposite mutation can exclude bulky substrates from binding to a specific protein.

Mutation that involve changes in the functional groups of the amino acids that make up an active site can provide more drastic changes than bulk tolerance or steric changes. Examples of many of these types of changes are available in the literature (see, eg: Recktenwald, A, et al, J. Biotech, 1993, 28, 1-23 and references cited therein; and Lesk, A, et al, "Antibody engineering: A Practical Guide", Ed. C. A K. Borrebaeck et al 1991, 1-75). In systems with high quality structural data, specific hydrogen bonding groups can be inserted to improve the interaction of polar substrates. Replacing uncharged residues with arginine, lysine, or histidine inserts groups that could interact with negatively charged substrates. Replacing residues with glutamic acid or aspartic acid can provide interaction with positively charged substrates and thus change the specificity of an enzyme substrate interaction.

In a further aspect of the present invention there is provided a recombinant conjugate comprising a tragetting antibody as hereinbefore described and a mutant enzyme of the present invention; such recombinant conjugate having been expressed from a single DNA construct or translated from a single RNA transcript. Also included within the scope of the present invention are the corresponding DNA and RNA molecules encoding such a conjugate. Preferred embodiments of such a conjugate reflect a combination of the preferred embodiments for its two component activities as hereinbefore expressed.

Such recombinant conjugates may be produced by expression of fusion constructs, generated by joining, in vitro, fragments of DNA that separately encode the enzyme and all or a part of a mAb that confers antigen specificity (Pastan, I et al, (1986) Cell, 47, 641-648). Such fusions may be constructed by standard techniques of recombinant molecular biology in ways that place the substrate-binding or active site of the polypeptide at the amino terminus or the carboxy terminus and preferably at the amino terminus. The mAb portion of the construct may be designed so that a single chain species of the antibody is connected to the enzyme portion either with the variable light or the variable heavy domain at the 5'-end. In either case, the variable light and variable heavy gene regions will be connected with a synthetic DNA fragment encoding a peptide linker that achieves the correct spatial arrangement of the antibody variable regions. Such a synthetic DNA region connecting the antibody portion of the fusion construct to the enzyme portion may also be desirable. In other forms, the gene encoding the enzyme may be fused to the 5'-end of the variable region of the heavy or light chain gene that extends to form a Fab or F(ab')₂ species. Other variations of the heavy chain gene may be used to form the enzyme/antibody recombinant conjugate. Such variations may include natural heavy chain isotypes, modified full-length heavy chain genes of all antibody isotypes, and heavy with various deletions, specifically, deletion of the C_(H2) domain may confer desirable pharmacokinetic properties. Alternatively, the fusion may produce a covalent bispecific antibody enzyme conjugate where one arm of the antibody is replaced by the enzyme in a fashion similar to but not limited to that described in De Sutter, K and Fiers, W. Mol Immunol 31 261 (1994), The term `conjugate` as used herein includes such recombinant conjugates.

Antibodies designed to have particular cellular specificities may be generated in any way well known in the art. Fragments of antibodies generated by recombinant DNA technology may also be used as targetting molecules.

Although the preferred antibodies for use will be those expressed from natural or recombinant human derived cell-lines, it is also possible to exploit antibodies from other antibody producing mammalian cell-lines providing such antibodies are not immunogenic to an extent liable to interfere significantly with therapy or that the antibodies have been `humanised` such that they no longer elicit an immune reaction in vivo. Such a humanized antibody may be a chimaeric antibody (Morrison et al P.N.A.S (1984), 81, 6851-6855; Boulianne et al, Nature, 1985, 314, 268-270 and Neuberger et al, Nature, 1985, 314, 268-70), or a CDR-grafted antibody (James et al, Nature, 1986, 321, 522-525Riechmann et al. Nature, 1988, 332, 323-327).

An antibody for use according to the present invention preferably is a monoclonal antibody or a fragment thereof. The antibody may therefore comprise a complete antibody, a (Fab')₂ fragment, a Fab' fragment, a Fab fragment, a light chain dimmer or a heavy chain dimmer or single chain species comprising the variable regions from heavy and light chains. The antibody may be an IgG such as IgG1, IgG2, IgG3, or IgG4; or IgM, IgA, IgE or IgD. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain.

The antibody may be a chimaeric antibody of the type described in WO 86/01533. A chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain and/or heavy chain variable domain. Typically the chimaeric antibody comprises both light and heavy chain variable domains. The non-immunoglobin region is fused to the C-terminus of the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and is preferably an enzyme region. The tow regions of the chimaeric antibody may be connected via a cleavable linker sequence.

Antibodies of any of the classes referred to above may be raised against any antigen characteristic of and essentially limited to, a particular target. Such targets include all types of human pathogens cells expressing antigens as a consequence of transformation or viral infection, cells expressing particular histocompatability antigens, cells involved in inflammatory responses, blood clots which may be targetted by antibodies against fibrin, etc.

Preferred antibodies for use according to the invention, the antigens which they target and the associated indication are detailed in FIG. 8.

Particularly preferred antibodies for use according to the invention include CAMPATH 1H®, ING-1c174, PR1.A3, 323/A3, G250, MOV18, 17-1A, NR-LU-10, U326, NR-CO-OZ2 as well as antibodies targetting mucins or carcinoembryonic antigen.

For the treatment of humans when the targetting moiety is an antibody, it is preferred to use and antibody which does not carry with it the risk of the development of an immune reaction against the antibody itself. Accordingly whilst it is possible to use mouse or rat non-clonal antibodies it is preferred to use antibodies which have been produced by recombinant DNA technology and which have been engineered to reduce the risk of causing an immune reaction. Thus it is possible to use a chimaeric antibody in which the constant domains of a mouse or rat antibody have been replaced by the constant domains of a human antibody. However, it is preferred to use a humanised or CDR-grafted antibody for the treatment of humans, i.e. an antibody in which the complementary determining regions from a mouse or rat antibody are combined with framework regions and constant domains from one or more human antibodies.

In one preferred embodiment the method according to the invention is carried out using the CDR-grafted antibody CAMPATH-1H (see Riechmann et al Nature, 322, 323-327 (1988)). As noted above, the antibody CAMPATH-1H can be produced in rate myeloma cells as originally described or it can be produced in ay other expression system, particularly an expression system suitable for the production of a correctly folded, glycosylated, mammalian protein. High yields of CAMPATH-1H have been obtained by expression in a genetically manipulated CHO cell line (Page and Sydenham, Biotechnology, 9, 64-68 (1991)).

The conjugation of the tragetting molecule to the enzyme may be carried out in any appropriate way which will neither interfere with the binding specificity and capability of the tragetting molecule nor inhibit the catalytic activity of the enzyme. In the case of proteinic tragetting molecules the conjugation may be carried out either by direct covalent linkage following the generation of reactive groups by treatment with protein modifying agents or by these of homo or heterobifunctional cross-linking molecules.

Commercially available linkers with amino or hydrazide groups may be reacted with aldehydes generated by oxidation of the sugar moieties of glycoproteins and carbohydrates to from a Schiff's base (Sela M. et al, Conjugates of Antibodies with Cytotoxic Drugs. Immunoconjugates Antibody Conjugates in Radioimaging and Therapy of Cancer (C. -W. Vogel, ed.) 189-216, (1987)). Alternatively linkers may be reacted with amino, carboxyl or sulfhydryl groups of the antibody or enzyme. (Wawrzynczak P. E. et al, Methods for Preparing Immunotoxins: Effect of the Linkage on Activity & Stability In : Immunoconjugates: Antibody Conjugates in Radioimaging and therapy of Cancer (C. -W. Vogel, ed.) 78-85(1987)).

Covalent linkage may be obtained by the generation of sulfhydryl groups and, particularly in the case of antibodies, by reduction of disulfide bonds. Such free sulfhydryl groups may be reacted with haloalkyl groups, p-mercuribenzoate groups and groups capable of Michael-type addition reactions, such as maleimides or groups as per (Mitra et al, J Amer Chem Soc, 101, 3097-3110, 1979).

Another method for linking enzyme to antibody comprises utilising the epsilon amino groups of lysine. Reactive succinimide esters, cyclic thio esters or anhydrides may be used to introduce carbonyl functions which may react with the lysine amino group or to introduce a thiol reactive group such as those discussed herein. Alternatively, carbonyl functions may be activated with carbodimides to form carboxamide bonds or the lysine amino group may be reacted with methyl amidate esters to form an amindinium bond.

One particular utility of the present invention is its application it in vitro or in vivo diagnostics. For example, in vitro detection of a particular antigen may be achieved by exposing a diagnostic sample to a conjugate of the present invention comprising a tragetting moiety such as an antibody capable of binding to the antigen and subsequent exposure to a prodrug such as a methotrexate prodrug of the present invention, which may be catalysed to methotrexate by the enzyme of the conjugate if antigen is present and conjugate is bound to the antigen. Prodrug such as MTX may be detected by standard HPLC analysis, or by the coupled spectrophotometric assay described herein.

In vivo diagnostic applications of the present invention include its ability to be used in tumour imaging. Particularly preferred conjugates for use in this application include those comprising antibodies to a tumour associated antigen and a mutant enzyme capable of catalysing to methotrexate an MTX-prodrug labelled in the benzene right with ¹³¹ I. Such a prodrug should be one which is otherwise refractory to endogenous catalysis. Conjugate and prodrug may be administered in a manner analogous to the therapeutic application of the invention and detection of the location of catalysed prodrug i.e. ¹³¹ I MTX achieved using standard equipment for detection of radioisotopic decay.

Salts of prodrugs of the present invention include pharmaceutically acceptable base salts, derived from an appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) slats, ammonium and NX+4 (wherein X is C₁₋₄ alkyl) salts. Suitable pharmaceutically acceptable salts also include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobrimic, sulfuric, nitric, phosphoric, maleic, salicylic, p-toluene-sulfonic, tartaric, citric, acetic, trifluoroacetic methanesulfonic, formic, succinic, naphthalene-2- sulfonic, isethionic, lactobionic and benzenesulfonic. The pharmaceutically acceptable prodrug salts may be prepared in conventional manner for example by treatment with the appropriate base or acid.

In a method of treatment according to the present invention the means of administration of conjugate and of prodrug will depend upon the disease and its severity and will be at the discretion of the practicing physician. Both the conjugate and prodrug may be administered by the conventional methods well known to those skilled in the art including intravenously, intraperitoneally, intralymphatically, subcutaneously, intradermally, intramuscularly, orally or in the case of a tumour by direct injection into the tumour. Both the prodrug and the conjugate will preferably be administered intravenously as a bolus or by continuous infusion. Preferably the dose of conjugate administered is in the rage 0.1 to 100 mg per kg of bodyweight per day and most preferably between 1.0 and 40 mg per kg of bodyweight per day. The preferred dose of the prodrug administered is in the range 0.01 to 100 mg per kg of bodyweight per day and preferably between 1.0 and 50 mg per kg of bodyweight per day.

It is preferred that the conjugate of targetting molecule and mutant enzyme be administered to the patient prior to administration of the prodrug. The period between administration of conjugate and prodrug must be sufficient to permit targetting of the conjugate to the targetted cells and effective removal from the system of excess conjugate which has not bound to the targetted cells. Unbound conjugate must be effectively removed to avoid activation of the subsequently administered prodrug by unbound conjugate present at sites distant from the target cells. The period between administration of conjugate and prodrug is preferably between 0.5 days and 7 days but may be reduced by use of any known technique for the accelerated clearance of such conjugates from the system; particularly by delivery of an effective amount of anti-enzyme antibody, which binds to the mutant enzyme and in turn accelerates elimination, or by extra corporeal circulation (Nilsson, I M, et al, Plasma ther, Transfus Technol, 1984, 5, 127; Wallmark, A, et al, Artificial Organs, 1984, 8, 72) involving removal of the unbound conjugate by methods such as passage through a column of an appropriate affinity matrix. It may also be desirable to couple galactosyl residues to the conjugate to facilitate unbound conjugate clearance via hepatocyte lectins.

Conditions susceptible to therapy according the present invention will clearly include those for which there exists a known chemotherapeutic agent and a corresponding prodrug thereof as hereinbefore defined and which presents pathology specific feature permitting binding of a conjugate of the present invention. Such conditions include neoplastic and non-neoplatic cellular transformation, autoimmune disease, inflammatory disease and viral, bacterial, fungal, mycoplasmal or parasitic infection.

a method of treatment according to the present invention may further comprise the co-administration of a number of agents capable of increasing the efficiency of the therapy. For example, interferons are known to increase the level of expression of certain potential ADEPT target antigens and therefore administration of an appropriate interferon before administration of conjugate may increase level of conjugate binding to target cells.

It is further preferred that a combination of a prodrug of a purine synthesis inhibitor such as an inhibitor of glycineamide ribonucleotide transformylase (GAR TFase) or a prodrug of a pyrimidine synthesis inhibitor such as an inhibitor of thymidine synthase (TS) be administered in combination with a prodrug of a dithydrofolate (DHFR) inhibitor as part of such therapy (Galivan, J. et al, J. Biol. Chem. 264, 10685, 1989Ferguson, K. et al Cancer Chemother. Pharmacol. 23, 173, 1989), It is further preferred that a combination of a prodrug of purine synthesis inhibitor such as an inhibitor of GAR TFase or a prodrug of a pyrimidine synthesis inhibitor such as an inhibitor of TS be administered in combination with DHFR inhibitor, or that a combination of a purine synthesis inhibitor such as an inhibitor of GAR TFase or a pyrimidine synthesis inhibitor such as an inhibitor of TS be administered in combination with a prodrug of a DHFR inhibitor as part of such therapy.

A further aspect of the present invention comprises a method for the potentiation of any antiviral compound, the efficacy of which may be augmented by elevation of intracellular kinase activity.

According to a preferred embodiment of this further aspect of the present invention, a conjugate of the present invention comprising a targetting molecule capable of directing conjugate to a virally-infected cell and a prodrug of a folate antagonist which may be one or more of a thymidylate synthase (TS) inhibitor, dihydrofolate reductase (DFHR) inhibitor or GAR transformylase inhibitor is used to deliver high does of folate antagonist directly to the virally-infected cells of a patient who is undergoing therapy. As viral infection is associated with increased levels of DNA synthesis, it is proposed that the delivery of an inhibitor of DNA synthesis in combination with such an antiviral may prove therapeutic as the inhibition of DNA synthesis will increase the level of intracellular kinases available for phosphorylating the antiviral compound.

In a preferred embodiment of this further aspect of the invention, the anti-viral is an anti-herpes compound selected from acyclovir(GB 1523865), valacyclovir a(EP308065), famiciclovir EP182024), penciclovir (EP141927), foscarnet (GB1585615), iododeoxyuridine, 5-bromovinyl-2'-deoxyuridine (GB1601020), arabino-furanosyl-5-bromovinyl-2'deoxyuridine (EP0031128), benzimidazole nucleosides, 1-(β-D-arabinofuranosyl)-5-(1-propynyl)uracil (EP272065) and 2'-deoxy-5- ethyl-β-4'-thiouridine (EP409575), or is an anti-HIV compound selected from 2',3'-dideoxyinosine (EP206497), 2,'3'-dideoxycytidine (J. Org. Chem 32(3) 817.818(1967)), (2R, 5S)-5- fluoro-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine (US5210085), (2R,5S)-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine(EP0382526), 5-chloro-2',3', -dideoxy-3'-fluorouridine (EP0305117) and (1S,4R)-4-(2amino-6-(cyclopropylamino)-9H-purin-9- yl)-2-cyclopentent-1-methanol (EP0434450) or is the anti-influenza compound 9-(2-Deoxy-2fluoro-β-D-ribofuranosyl) guanine) (EP0417999).

In the most preferred embodiment of this further aspect of the invention, the antiviral compound is an anti-HIV compound such as zidovudine (EP196185), (Retrovir: registered trade mark of The Wellcome Foundation Limited), the conjugate comprises an antibody against an HIV-associated antigen such as GP210 and the prodrug is a prodrug of a TS inhibitor.

The present invention provides for the use of a novel prodrug of the present invention as hereinbefore defined, or a pharmaceutically acceptable, salt, solvate, N-oxide or physiologically functional derivative thereof, in therapy or in the preparation of a medicament for use in the treatment of any of the conditions hereinbefore described.

Also provided is the use of a mutant enzyme of the present invention, or a conjugate thereof as hereinbefore described, in therapy or in the preparation of medicament for use in the treatment of any of the conditions hereinbefore described.

Pharmaceutical formulations for mutant enzyme, antibody and prodrug may each include but are not limited to solutions, suspensions, tablets, powders, and other formulations known to those skilled in the art. The formulations may contain pharmaceutically acceptable stabilisers and carriers including human serum albumin or other proteins, pH buffering agents, and physiological or other nontoxic salts as well as non toxic solubilisers known to those skilled in the art. Preferably a pharmaceutical formulation of mutant enzyme and antibody will comprise conjugate in a sterile solution. A pharmaceutical formulation of prodrug will preferably comprise a sterile solution or a solid.

A yet further aspect of the present invention comprises a kit comprising a pharmaceutical formulation of conjugate and a pharmaceutical formulation of prodrug which may be converted to drug by the conjugate.

A yet further aspect of the present invention comprises the combination of a prodrug and a conjugate of the present invention as hereinbefore defined.

A yet further aspect of the present invention comprises the use of a novel prodrug as hereinbefore defined but excluding the previously stated proviso for use in the preparation of a medicament for use in the treatment of any of the conditions hereinbefore described.

A yet further aspect of the present invention comprises the use of a novel prodrug as hereinbefore defined but excluding the previously stated proviso, for use in the treatment of any of the conditions hereinbefore described.

A yet further aspect of the present invention comprises the use of a prodrug in the preparation of a medicament for use in the treatment of a patient who has previously been administered with a conjugate of the present invention as hereinbefore defined.

A further aspect of the invention comprises the cloned version of human carboxypeptidase A2 described herein as well as the DNA encoding carboxypeptidase A2.

A further aspect of the invention comprises any DNA sequence encoding a mutant enzyme for use according to the present invention and plasmids, expression vectors, and cell lines containing such DNA. Preferably such DNA encodes mutant carboxypeptidase A1 or A2 mutants as herein described and most preferable mutant carboxypeptidase A1 or A2 wherein position 268 is glycine.

A further aspect of the invention comprises any DNA sequence encoding a mutant enzyme for use according to the present invention and plasmids, expression vectors, and cell lines containing such DNA. Preferably such DNA encodes mutant carboxypeptidase A1 or A2 mutants as herein described and most preferable mutant carboxypeptidase A1 or A2 wherein position 268 is glycine.

A further aspect of the invention comprises DNA sequence encoding a conjugate according to the present invention and plasmids, expression vectors, and cell lines containing such DNA.

A yet further agreed aspect of the invention is a method of converting a prodrug to a drug which is cytotoxic to cells.

All references to alternatives herein, such as alternative patterns of chemical substitutions, shall be construed as inclusive and not as exclusive.

As used herein the term "halo" means fluoro, chloro, bromo or iodo.

The terms "aryl" means phenyl, naphthyl or anthracenyl optionally substituted by one or more halo, hydroxy, nitro, cyano, trifluoromethyl, C₁₋₆ alkyl, C₁₋₆ alkoxy or amino optionally substituted with C₁₋₆ alkyl.

The term "heteroaryl" means an aromatic monocyclic or bicyclic fused ring system comprising 5 to 10 carbon atoms wherein one or more ring atoms are independently selected from nitrogen, oxygen or suffer.

"Carboxamide" groups can be unsubsituted or substituted C₁₋₆ alkyl.

A mutant mammalian enzyme as used herein shall be taken to be any enzyme with a sequence differing by at least one amino acid from the amino acid sequence or sequences of that enzyme in the mammal to which the therapy is applied. Preferably the mammal to which the therapy is applied is a human patient and preferably the amino acid sequence of the mutant mammalian enzyme does not differ by more than 15% from the amino acid sequence or sequences of that enzyme in the mammal to which the therapy is applied.

All references hereinbefore and in the Claims hereinafter to residue numbers of amino acids relate to the residue number indicated in the Tables contained herein and not to the residue numbers given in the Sequence Listing herein which follows a different numbering convention.

EXPERIMENTAL PROCEDURES General Comments

All solvents were reagent grade and were used without further purification. Anhydrous solvents were dispensed from Aldrich Sure Seal bottles using a dry syringe. Chemicals are reagent grade and were used without purification. The full name and address of the suppliers of the reagents is given when first cited. Thereafter, an abbreviated name is used.

¹ H-NMR spectra were obtained using Varian XL-200 or XL-300 spectrometers at 200 and 300 MHz respectively. Chemical shifts are expressed in ppm downfield from tetramethylsilane. Spectral data are tabulated in order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), number of hydrogens, coupling constant(s) in Hertz, descriptor. Chemical ionization (CI) mass spectra were performed by Oneida Research Services, Inc. Whitesboro, N.Y. 13492. Atmospheric pressure chemical ionization (APCI) mass spectra were obtained using a Fisons platform mass spectrometer. Fast atom bombardment (FAB) mass spectra were obtained using a VG Analytical 70SQ spectrometer. Ion spray mass spectra were obtained using a Sciex API III spectrometer. Elemental analysis were performed by Atlantic Microlabs, Inc., Atlanta, Ga. 30366. Reverse analyses phase analytical HPLC's were run on C18 analytical columns using a Waters HPLC system consisting of a 600E System Controller, a 715 UltraWisp sample processor, and a 991 Photodiode Array Detector. Semi-preparative HPLC's were run on a Regis column (C18, Prep-100-60-ODS-FEC, 10 mm packing, 25 cm×21.1 mm i.d.) using a similar system equipped with a manual injector.

Abbreviations used are: grams (g), milliliters (mL), liters (L), hours (h), minutes (min), room temperature (RT), calculated (calcd.), decomposition (dec.), molecular weight (mw), tetrahydrofuran (THF), dimethyl formamide (DMF), dimethyl sulphoxide (DMSO), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC), dicyclohexylcarbdiimide (DCC), diethyl cyanophosphonate (DECP), (benzyloxy)carbonyl (Cbz), and trifluoroacetic acid (TFA).

Some examples prepared were very tenacious of water and/or other hydroxylic solvents (e.g. DMF), and/or were obtained as whole or partial salts of organic or inorganic acids (e.g. trifluoroacetic acid, hydrochloric acid). Such addends are indicated in the analytical data for appropriate examples; although these addends may not be indicated as part of the compound name, their presence in the isolated products is noted by the inclusion of salt and hydration data in the molecular formulas and analytical data in appropriate examples.

EXAMPLE 1 2-Fluoro-4-nitrobenzoic Acid

A 3L 3-necked flask equipped with a magnetic stirrer was charged with 25.00 g of 2-fluoro-4-nitrotoluene (Aldrich Chemical Co., Milwaukee, Wis., 53233), 1.65 L of 1 N NaOH, and 25.00 g of KMnO₄. The resulting mixture was heated to 95° C. with stirring. Additional 25.00 g portions of KMnO₄ were added after 1 h and 2 h. After stirring at 95° C. for an additional hour the reaction mixture was cooled to RT and filtered through celite to remove MnO₂. The filtrate was concentrated in vacuo to 500 mL and acidified to pH 2 by addition of concentrated H₂ SO₄. A yellow-orange precipitate formed which was collected by vacuum filtration. The crude solid was dissolved in 300 mL of 1 N aqueous NaOH, acidified to pH 2 and the mixture extracted with CH₂ Cl₂ (4×150 mL). The combined CH₂ Cl₂ extracts were washed with 0.1 N aqueous HCl (2×100 mL), dried over anhydrous Na₂ SO₄, and concentrated in vacuo to afford 12.44 g (42%) of 2-fluoro-4-nitrobenzoic acid as a yellow crystalline solid, m.p. 165-167° C.

EXAMPLE 2 Ethyl 2-fluoro-4-nitrobenzoate

To a dry 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 3.00 g of 2-fluoro-4-nitrobenzoic acid, 3.00 g of NaHCO₃, and 20 mL of anhydrous dimethylacetamide. The suspension was treated with 3.50 mL of ethyl iodide and stirred at RT under N₂ for 18 h. The reaction mixture was filtered to remove solids and the filtrate concentrated in vacuo to give an orange residue which was dissolved in 100 mL of CH₂ Cl₂. The solution was washed with saturated aqueous NaHCO₃ (3×50 mL), water (1×60 mL), and dried over anhydrous Na₂ SO₄. Removal of drying agent by filtration and passage through a silica gel plus (3×5 cm) gave a light yellow solution which was concentrated in vacuo to afford 3.10 g (90%) of ethyl 2-fluoro-4-nitrobenzoate as a light yellow crystalline solid, m.p. 70-73° C.

EXAMPLE 3 Ethyl 4-amino-2-fluorobenzoate

To a 250 mL round bottomed flask equipped with a magnetic stirrer were added 3.00 g of ethyl 2-fluoro-4-nitro-benzoate and 100 mL of MeOH. The solution was deoxygenated by bubbling nitrogen through for 10 min and then treated with 2.00 g of Raney nickel (Raney 3200, Davison Chemical, Chattanooga, Tenn. 37406) which had been washed with water and EtOH. The solution was subjected to hydrogenation at 1 atm with stirring for 40 min. The flask was purged with nitrogen and the catalyst was removed by filtration. The filtrate was concentrated in vacuo to afford 2.40 g (93%) of ethyl-4-amino-2-fluorobenzoate as a tan crystalline solid, m.p. 110-112° C.

EXAMPLE 4 9-Bromomethyl-3-methylbenzo(f)quinazolin-1(2H)-one

To a 3L 3-necked flask equipped with a magnetic stirrer, a nitrogen inlet, and a reflux condenser were added 1.4 L of 1,2-dichloroethane. The solvent was heated to reflux and 10.00 g of 3,9-dimethylbenzo(f)-quinazolin-1(2H)-one (prepared according to the method described in International Patent Application WO 91/19700) were slowly added. This was followed by addition of 8.73 g of N-bromosuccinimide (Aldrich) and 1.00 g of benzoyl peroxide (Aldrich). After heating at reflux for 2 h the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was slurried with 60 mL of EtOH and the solid was collected by vacuum filtration. The product was washed with 50 mL of EtOH and dried in vacuo to afford 12.39 g of a light tan solid, m.p.>200° C. Analysis by ¹ H-NMR indicated a mixture consisting of 80% of 9-bromomethyl-3-methylbenzo(f)quinazolin-1(2H)-one and 20% of starting material. The material was carried on to the next step without further purification.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 9.86 (s,1H, aromatic CH), 8.28 (d, 1H, J=9.0, aromatic CH), 8.06 (d, 1H, J=8.4, aromatic CH), 7.72 (d, 1H, J=8.2, aromatic CH), 7.65 (d, 1H, J=8.8, aromatic CH), 4.94 (s, 2H, ArCH₂ Br), 2.49 (s, 3H, CH₃).

EXAMPLE 5 Ethyl 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoate

To a 500 mL 3-neck flask equipped with a magnetic stirrer, a nitrogen inlet, and a reflux condenser were added 5.00 g of 9-bromomethyl-3-methylbenzo(f)quinazolin-1(2H)-one, 3.02 g of ethyl 4-amino-2-fluorobenzoate, 4.17 g of NaHCO₃, and 42 mL of anhydrous DMF. The reaction mixture was heated to 100° C. with stirring under nitrogen for 1.5 h.

After cooling to RT the mixture was filtered to remove solids and the crude product was deposited on 30 g of silicon gel by concentrating the filtrate in vacuo in the presence of silica gel. The product/silica gel mixture was applied to a column (360 g SiO₂) and purified by flash chromatography using MeOH--EtOAc gradient elution (EtOAc→2% MeOH--EtOAc). This afforded 3.50 g (65%) of ethyl 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoate as a white powder. m.p. >200° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.54 (s,1H, NH), 9.85 (s, 1H, aromatic CH), 8.23 (d, 1H, J=8.7, aromatic CH), 8.01 (d, 1H, J=8.2, aromatic CH), 7.69-7.50 (m, 3H, aromatic CH), 6.52 (dd, 1H, J=1.8, 8.9; aromatic CH), 6.40 (dd; 1H; J=1.7, 14.7; aromatic CH), 4.58 (d, 2H, J=5.6, ArCH₂ N), 4.19 (q, 2H, J=7.0, ethyl CH₂), 2.44 (s, 3H, CH₃), 1.24 (t, 3H, J=7.0, ethyl CH₃).

EXAMPLE 6 4-(((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid

A 100 mL 3-necked flask equipped with a magnetic stirrer was charged with 0.200 g of ethyl 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoate, 18 mL of 1 N NaOH, and 20 mL of EtOH. After stirring overnight at RT, the solution was concentrated to 20 mL in vacuo and acidified to pH 2.5 by addition of concentrated HCl. The resulting precipitate was collected by vacuum filtration, washed with 15 mL of water, and dried in vacuo to afford 0.15 g (77%) of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid as a white powder, m.p. >250° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.20-12.30 (br s, 1H, COOH), 9.83 (s, 1H, aromatic CH), 8.24 (d, 1H, J=8.6, aromatic CH), 8.06 (d,1H, J=8.4, aromatic CH), 7.80-7.40 (m, 4H, aromatic CH), 6.51 (d, 1H, J=7.9, aromatic CH), 6.37 (d, 1H, J=14.2, aromatic CH), 4.60 (s, 2H, ArCH₂ N), 2.48 (s, 3H, CH₃).

EXAMPLE 7 N-((Benzyloxy)carbonyl)-5-O-ethyl-L-glutamic Acid

A 2L 3-necked flask equipped with a magnetic stirrer and a thermometer was charged with 25.0 g of glutamic acid γ-ethyl ester (Sigma Chemical Co., St. Louis, Mo., 63178) and 1 L of distilled water. The solution was heated to 60° C. with stirring and treated with 30 g of NaHCO₃ followed by 25 mL of benzyl chloroformate (Aldrich). The flask was allowed to cool to RT and stirring was continued for 2 h. The mixture was extracted with ethyl ether (5×100 mL) and the ether extracts were discarded. The aqueous phase was acidified to a congo red end point by addition of concentrated aqueous HCl and the resulting emulsion was extracted with CH₂ Cl₂ (5×100 mL). The combined CH₂ Cl₂ extracts were washed with 0.05 N aqueous HCl (2×100 mL), dried over anhydrous Na₂ SO₄, and concentrated in vacuo to a volume of 50 mL. The solution was chilled in an ice water bath and triturated with addition of 200 mL of pentane. The resulting solid was collected by vacuum filtration and dried in vacuo to afford 37.0 g (83%) of N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutamic acid as a white powder, m.p. 82-84° C.

EXAMPLE 8 Ethyl 3-(1-naphthyl)-L-alaninate

To a 250 mL 3-necked flask equipped with a magnetic stirrer and a reflux condenser were added 3.00 g of 3-(1'-naphthyl)-L-alanine (Schweizerhall, Inc., Piscataway, N.J., 08854) and 75 mL of absolute EtOH. The suspension was acidified by bubbling HCl gas through until a clear solution was obtained and the solution was heated at reflux with stirring for 5 h. The solution was cooled to RT and concentrated in vacuo to give a white solid, which was suspended in 100 mL of 5% aqueous NaHCO₃ and extracted with EtOAc (3×60 mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO₃ (3×50 mL), water (1×50 mL), and dried over anhydrous Na₂ SO₄. The drying agent was removed by filtration and the filtrate concentrated in vacuo to give an oil which was dissolved in 40 mL of ethyl ether. The ether solution was treated with 13.9 mL of 1 N ethereal HCl and the resulting precipitate was collected by vacuum filtration and dried in vacuo to afford 3.70 g (95%) of ethyl 3-(1-naphthyl)-L-alaninate HCl as a white powder, m.p. 182-184° C.

EXAMPLE 9 Ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate

A 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 3.70 g of ethyl 3-(1-naphthyl)-L-alaninate HCl, 4.09 g of N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutamic acid, 60 mL of CH₂ Cl₂, and 1.45 mL of N-methylmorpholine. The mixture was chilled to 0° C. in an ice bath, treated with 2.66 g of EDC (Aldrich) and stirred under nitrogen. After 1 h at 0° C. the solution was allowed to warm to RT and stirring was continued for an additional 3 h. The solution was concentrated in vacuo and the residue was dissolved in 100 mL of EtOAc. The EtOAc solution was washed with 10% aqueous citric acid (3×50 mL) followed by 5% aqueous NaHCO₃ (3×50 mL). The solution was dried over anhydrous Na₂ SO₄, concentrated in vacuo to 20 mL and triturated with addition of 50 mL of pentane. The resulting solid was collected by vacuum filtration and dried in vacuo to afford 5.80 g (82%) of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate as a white powder, m.p. 124-126° C.

EXAMPLE 10 Ethyl 5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate

A 500 mL Parr bottle was charged with 0.19 g of 10% Pd(C) and 60 mL of absolute EtOH under nitrogen. The catalyst-solvent mixture was deoxygenated by bubbling nitrogen through for 5 minutes and then treated with 0.14 mL of acetyl chloride followed by 1.02 g of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate. The mixture was hydrogenated at 45 psi for 18 h. The bottle was purged with nitrogen and the catalyst was removed by filtration through celite. The filtrate was concentrated in vacuo to afford 0.81 g (97%) of ethyl 5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate HCl as a light yellow foam, m.p. 85-90° C. (dec.).

EXAMPLE 11 Ethyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam-1-yl)-3-(1-naphthyl)-L-alaninate

A 100 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 0.50 g of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid and 10 mL of DMPU (Aldrich). The mixture was alternately heated and sonicated to dissolve the solid and the resulting solution was stirred under nitrogen in the presence of 0.50 g of 3 Å molecular sieves for 18 h. The mixture was cooled to 0° C. in an ice bath and treated with 0.15 mL of N-methylmorpholine followed by 0.17 mL of isobutyl chloroformate. After stirring at 0° C. for 15 min the reaction mixture was treated with a solution of 0.69 g of ethyl 5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate HCl and 0.17 mL of N-methylmorpholine in 4 mL of anhydrous DMF. The reaction was maintained at 0° C. for 1 h and was then allowed to warm to RT and stirred for an additional 4 h. The mixture was filtered to remove solids and the filtrate was mixed with 150 mL of water. The resulting crude solid was collected by filtration, dried in vacuo, and subjected to flash chromatography on 200 g of silica gel (1% MeOH/EtOAc→5% MeOH/EtOAc) to afford 0.40 g (40%) of ethyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam-1-yl)-3-(1-naphthyl)-L-alaninate as a white powder, m.p. 225° C. (dec.).

¹ H-NMR: (200 MHz, DMSO-d₆)δ 12.55 (s,1H, benzaquinazoline NH), 9.87 (s, 1H, aromatic CH), 8.63 (d, 1H, J=7.5, amide NH), 8.24 (d, 1H, J=8.3, aromatic CH), 8.09 (d, 1H, J=7.7, aromatic CH), 8.03 (d, 1H, J=8.0, aromatic CH), 7.92 (d, 1H, J=7.1, aromatic CH), 7.79 (t, 1H, J=5.6, 4-aminobenzoyl NH), 7.68-7.30 (m, 9H, aromatic CH, amide NH), 6.56 (d, 1H, J=7.9, aromatic CH), 6.42 (d, 1H, J=14.4, aromatic CH), 4.68-4.43 (m, 4H, benzoquinazoline-9-CH₂, glu methine, naphthylala methine), 4.10-3.92 (m, 4H, ethyl CH₂), 3.63-3.30 (m, 2H, naphthyl-CH₂), 2.45 (benzoquinazoline CH₃), 2.29 (m, 2H, glu 4-CH₂), 2.07-1.74 (m, 2H, glu 3-CH₂), 1.14(t, 3H, J=7.1, ethyl CH₃), 1.03 (t, 3H, J=7.1, ethyl CH₃).

EXAMPLE 12 N-(N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl)-3-(1-naphthyl)-L-alanine

To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.15 g of ethyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam-1-yl)-3-(1-naphthyl)-L-alaninate, 8 mL of 1:1 THF:H₂ O, and 20 mg of LiOH.H₂ O. The mixture was stirred at RT under nitrogen. After 1 h analysis of the solution by tlc (SiO₂, 3% MeOH/CH₂ Cl₂) showed no remaining starting material and a new component at R_(f) =0.0. The solution was acidified to pH 5 by addition of 1 N aqueous HCl and the resulting white suspension was concentrated in vacuo to dryness. Analysis of the residue by reverse phase HPLC (C18, 65:35:0.1 H₂ O:MeCN:TFA) indicated a major component (95%) at k'=3.17 and a minor component (5%) at K'=4.90. The crude product was purified by semi-preparative reverse phase HPLC using gradient elution (C18, 70:30:0.1 .ae butted.65:35:0.1 H₂ O:MeCN:TFA over 10 min). Pure fractions (as determined by analytical HPLC) were combined, concentrated to 20 mL in vacuo and lyophilized to afford 76 mg (50%) of N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)-quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl)-3-(1-naphthyl)-L-alanine as an off white powder, m.p. 180° C. (dec.).

HPLC: one peak on C18, K'=3.53, 65:35:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 13.30-12.20 (br m, COOH), 9.82 (s,1H, aromatic CH), 8.44 (d, 1H, J=8.1, amide NH), 8.26 (d,1H, J=8.9, aromatic CH), 8.11 (d, 1H, J=8.3, aromatic CH), 8.03 (d, 1H, J=8.3, aromatic CH), 7.84 (d, 1H, J=8.2, aromatic CH), 7.73 (d, 1H, J=7.8, aromatic CH) 7.67-7.27 (m, 9H, aromatic CH, amide NH), 6.53 (d, 1H, J=8.5, aromatic CH), 6.38 (d, 1H, J=14.7, aromatic CH), 4.63-4.38 (m, 4H, benzoquinazoline-9-CH₂, glu methine, naphthylala methine), 3.55 (m, 1H, naphthyl-CH₂), 3.27 (m, 1H, naphthyl-CH₂), 2.43 (s, 3H, benzoquinazoline CH₃), 2.18 (m, 2H, glu 4-CH₂), 2.00-1.70 (m, 2H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₉ H₃₄ N₅ O₇ F.1.5H₂ O.0.5TFA (MW 787.76): C, 60.99; H, 4.80; N, 8.89. Found: C, 60.96; H, 4.82; N, 8.98

Mass Spectrum: (FAB) 704(M+H)⁺, 613, 489, 371, 309.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 223.4 (82600), 266.4 (53100), 348.6 (5500). λ_(min) (ε) 243.2 (23500), 340.2 (3430).

EXAMPLE 13 Ethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate

To a solution of 0.24 mL of DECP (Aldrich) and 0.22 mL of Et₃ N in 20 mL of anhydrous DMF in a 100 mL 3-necked flask were added 0.20 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich Chemical Co., Milwaukee Wis., 53233) and 0.04 mL of Et₃ N dissolved in 3 mL of DMPU. The solution was stirred at RT under nitrogen for 3 h and treated with a solution of 0.23 g of ethyl 5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate HCl and 0.15 mL of Et₃ N in 2 mL of anhydrous DMF. After stirring for 70 h the reaction mixture was concentrated in vacuo to 3 mL and treated with 60 mL of ethyl ether to precipitate the crude product as a tacky orange solid. The ether was decanted and the product dissolved in 60 mL of CHCl₃. The solution was extracted with 1% aqueous NH₄ OH, dried over anhydrous Na₂ SO₄ and concentrated in vacuo to dryness. The yellow-orange residue was purified by flash chromatography on 150 g of silica gel (7:7:1 CH₂ Cl₂ : acetone:MeOH) to afford 0.26 g (70%) of ethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate as a yellow powder, m.p. 135° C. (dec).

¹ H-NMR: (200 MHz, DMSO-d₆)δ 8.59 (s,1H, pteridinyl-7-CH), 8.45 (d, 1H, J=7.2, amide NH), 8.10 (d, 1H, J=8.0, aromatic CH), 8.04 (d, 1H, J=8.2, aromatic CH), 7.92 (d, 1H, J=6.8, aromatic CH), 7.83-7.45 (m, 6H, aromatic CH, NH₂), 7.41-7.23 (m, 2H, aromatic CH), 6.84 (d, 2H, J=8.8, aromatic CH), 6.65 (br s, 2H, NH₂), 4.81 (s, 2H, pteridinyl-CH₂), 4.63-4.39 (m, 2H, glu methine, naphthylala methine), 4.13-3.91 (m, 4H, ethyl CH₂), 3.63-3.32 (m, 2H, naphthyl-CH₂), 3.24 (s, 3H, N--CH₃), 2.34 (t, 2H, J=7.6, glu-4-CH₂), 2.10-1.80 (m, 2H, glu-3-CH₂), 1.17 (t, 3H, J=7.1, ethyl-CH₃), 1.03 (t, 3H, J=7.1, ethyl-CH₃).

EXAMPLE 14 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-(1-naphthyl)-L-alanine

To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.13 g of ethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-3-(1-naphthyl)-L-alaninate, 10 mL of EtOH and 15 mL of 0.2 N NaOH. The resulting solution was stirred at RT under N₂ for 2.5 h, acidified to pH 5 by addition of 1 N aqueous HCl, and concentrated in vacuo to dryness. Analysis of the yellow solid by reverse phase HPLC (C18, 70:30:0.1 H₂ O:MeCN:TFA) indicated a major component (93%) at K'=4.3 and four minor components at K'=6.8, 3.5, 2.9 and 1.9. A 70 mg portion of the crude product was purified by semi-preparative reverse phase HPLC (C18, 70:30:0.1 H₂ O:MeCN:TFA). Pure fractions (as determined by analytical HPLC) were combined, concentrated in vacuo to 20 mL and lyophilized to afford 36 mg (53%) of N-4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-(1-naphthyl)-L-alanine as a yellow powder, m.p. 175° (dec).

HPLC: one peak on C18, K'=3.23, 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 13.00-11.95 (br m, 2H, COOH), 8.65 (s, 1H, pteridinyl-7-CH), 8.45 (br, 1H, NH₂), 8.36-8.18 (br, 1H, NH₂), 8.25 (d, 1H, J=7.5, amide NH), 8.10 (d, 1H, J=8.6, aromatic CH), 7.96 (d, 1H, J=7.8, aromatic CH), 7.90 (d, 1H, J=7.7, aromatic CH), 7.70 (m, 3H, aromatic CH), 7.60-7.45 (m, 3H, aromatic CH, NH₂), 7.39-7.15 (m, 3H, aromatic CH, amide NH, NH₂), 6.81 (d, 2H, J=8.6, aromatic CH, 4.84 (s, 2H, pteridinyl-CH₂), 4.51 (m, 1H, naphthylala methine), 4.42 (m, 1H, glu methine), 3.57 (m, 1H, naphthylala-CH₂), 3.28 (m, overlapping H₂ O peak, naphthylala CH₂), 3.25 (s, 3H, N--CH₃), 2.23 (t, 2H, J=7.5, glu-4-CH₂), 2.02-1.72 (m, 2H, glu-3-CH₂).

Elemental Analysis: Calcd. for C₃₃ H₃₃ N₉ O₆.1.5 H₂ O.0.5 TFA (MW 735.72): C, 55.51; H, 5.00; N, 17.13. Found: C, 55.65; H, 5.09; N, 16.92.

Mass Spectrum: (FAB) 652 (M+H)⁺, 581, 461, 371, 309.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 224.3 (74700), 260.3 (24900), 295.9 (24100), 372.5 (7150) λ_(min) (ε) 242.3 (15200), 271.0 (20500), 345.2 (5550).

EXAMPLE 15 N-((Benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid

To a 1 L 3-necked flask equipped with a thermometer and a magnetic stirrer were added 4.90 g of L-glutamic acid γ-methyl ester (Sigma) and 200 mL of water. The solution was heated to 60° C., treated with 6.40 g of NaHCO₃ followed by 5.20 mL of benzyl chloroformate (Aldrich) and allowed to cool to RT with vigorous stirring. After 4 h the reaction mixture was extracted with ethyl ether (4×100 mL) and the ether extracts were discarded. The aqueous solution was acidified to a congo red endpoint by addition of concentrated HCl. An oily emulsion resulted which was extracted with CH₂ Cl₂ (4×60 mL). The combined organic extracts were washed with 0.05 N aqueous HCl (2×60 mL), dried over anhydrous Na₂ SO₄ and concentrated in vacuo to afford 8.23 g (92%) of N-((benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid as a white crystalline solid, m.p. 61-63° C.

EXAMPLE 16 Glycine Tert-butyl Ester-benzophenone Imine

A 500 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 10.00 g of glycine-tert-butyl ester HCl (Sigma) and 200 mL of anhydrous CH₂ Cl₂. The solution was treated with 10.80 g of benzophenone imine (Aldrich) and stirred at RT under nitrogen for 24 h. A fine white precipitate of NH₄ Cl formed during the reaction. The mixture was concentrated in vacuo to dryness to give a white solid which was suspended in 250 mL of ethyl ether and washed with water (3×200 mL) to remove NH₄ Cl. The ether solution was dried over anhydrous MgSO₄ and concentrated in vacuo to afford a white solid. The material was dissolved in 30 mL of CH₂ Cl₂ and triturated with addition of 150 mL of pertane. A white crystalline solid resulted which was collected by filtration, dried in vacuo, and identified as glycine tert-butyl ester-benzophenone imine by ¹ H-NMR yield=15.0 g (85%), m.p. 110-112° C.

EXAMPLE 17 2-Iodobenzyl Bromide

To a dry 100 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer were added 5.00 g of 2-iodobenzyl alcohol (Aldrich) and 20 mL of anhydrous CH₂ Cl₂. The resulting suspension was cooled to 0° C. and treated with 0.71 mL of PBr₃ (Aldrich) by dropwise addition over a 2 minute period. The solid slowly dissolved giving a light yellow solution which was stirred under nitrogen at 0° C. for 2 h and then allowed to warm to RT with continued stirring for 18 h. The solution was concentrated in vacuo to dryness and the residue was dissolved in 60 mL of ethyl ether. The ether solution was washed with ice cold 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous Na₂ SO₄, and concentrated in vacuo to afford 5.65 g (89%) of 2-iodobenzyl bromide as a white crystalline solid, m.p. 52-54° C.

EXAMPLE 18 2-Iodo-L-phenylalanine tert-butylester-benzophenone imine

A 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 20.00 g of glycine tert-butyl ester-benzophenone imine, 24.13 g of 2-iodobenzyl bromide, 2.85 g of N-benzylcinchonidinium chloride (Fluka Chemical Corp., Ronkonkoma, N.Y., 11779), and 105 mL of CH₂ Cl₂. The resulting solution was treated with 110 mL of 50% (W/W) aqueous NaOH and the mixture was stirred vigorously at RT under nitrogen. After 24 h, tlc (silica gel, 7:1 hexane:EtOAc) indicated a major new component at R_(f) =0.65, unreacted starting material at R_(f) 32 0.85 and three minor components at R_(f) =0.20-0.45. The CH₂ Cl₂ phase was separated, washed with water (3×70 mL), dried over anhydrous Na₂ SO₄ and concentrated in vacuo to afford a thick pink oil. The crude product was subjected to flash chromatography on 550 g of silica gel eluting with 7:1 hexane:EtOAc (column was pretreated by passing through two column volumes of 0.5% Et₃ N in 7:1 hexane:EtOAc) to give 26.4 g (76%) of the desired enantiomer mixture as identified by ¹ H-NMR. The product was determine to by a mixture of two enantiomers by chiral HPLC (Pirkle phenylglycine, 99:1 hexane: isopropanol, flow rate=1 mL/min): K'=3.66 (76%), K'=3.28 (24%). The mixture was dissolved in 160 mL of hexane and allowed to crystallize at 4° C. for 2 days. The solid was separated by filtration and dried in vacuo to afford 10.72 g (31%) of racemic material. The filtrate was concentrated in vacuo to give 15.72 g (45%) of enantioenriched material (2-iodo-L-phenylalanine tert-butyl ester-benzophenone imine, ee=86%) as a transparent, viscous oil. The product was used in subsequent steps without further purification.

¹ H-NMR: (200 MHz, CDCl₃) d 7.72 (d, 1H, J=7.8, aromatic CH), 7.60 (m, 2H, aromatic CH), 7.41-7.21 (m, 6H, aromatic CH), 7.19 (m, 2H, aromatic CH), 6.86 (m, 1H, aromatic CH), 6.55 (d, 2H, J=6.6, aromatic CH), 4.34 (dd; 1H; J=3.9, 9.6; methine), 3.48-3.21 (m, 2H, Ar--CH₂), 1.47 (s, 9H, t--Bu).

Mass Spectrum: (Cl, CH₄) 512 (M+H)⁺, 484, 456, 410, 384, 217.

EXAMPLE 19 2Iodo-L-phenylalanine

A 500 mL round bottomed flask equipped with a reflux condenser and a magnetic stirrer was charged with 15.72 g of 2-iodo-L-phenylalanine tert-butyl ester-benzophenone imine (86% ee) and 165 mL of 6 N aqueous HCl. The mixture was heated at reflux with stirring for 4 h and cooled to RT to afford a mixture of white crystalline solid and an immicible organic phase (benzophenone). The mixture was partitioned between water (100 mL) and ethyl ether (70 mL). The aqueous phase was separated, washed with four additional 70 mL portions of ether and concentrated in vacuo to dryness to give 9.5 g (94%) of 2-iodo-L-phenylalanine HCl as a fluffy white solid, m.p. 240° C. (dec).

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.90-8.23 (br s, 3H, --NH₃ ⁺), 7.88 (d, 1H, J=8.1, aromatic CH), 7.39 (m, 2H, aromatic CH), 7.04 (m, 1H, aromatic CH), 4.06 (t, 1H, J=7.4, methine), 3.23 (m, 2H, Ar--CH₂).

EXAMPLE 20 Methyl 2-iodo-L-phenylalaninate

To a 500 mL 3-necked flask equipped with a magnetic stirrer and a reflux condenser were added 5.00 g of 2-iodo-L-phenylalanine HCl and 100 mL of anhydrous MeOH. The mixture was stirred and acidified by bubbling HCl gas through for 3 min. A clear solution resulted which was heated at reflux for 5 h. The solution was cooled to RT and concentrated in vacuo to dryness to give a white solid. The material was suspended in 120 mL of CH₂ Cl₂ and washed with 5% aqueous NaHCO₃ (4×80 mL). The CH₂ Cl₂ solution was concentrated to a volume of 20 mL, diluted to 60 mL with ethyl ether and treated with 16 mL ethereal HCl. A white solid precipitated which was collected by vacuum filtration and dried in vacuo to afford 4.90 g (94%) of methyl 2-iodo-L-phenylalaninate HCl, m.p. 194-196° C.

EXAMPLE 21 Methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-2-iodo-L-phenylalaninate

A dry 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 3.00 g of methyl 2-iodo-L-phenylalaninate HCl, 2.59 g of N-((benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid and 80 mL of CH₂ Cl₂. The mixture was cooled to 0° C. in an ice water bath and was treated with 0.97 mL of N-methylmorpholine followed by 1.77 g of EDC (Aldrich). The reaction mixture was stirred at 0° C. for 1 h and allowed to warm to RT with continued stirring for an additional 3 h. The solution was diluted with 100 mL of CH₂ Cl₂, washed with 5% aqueous citric acid (3×70 mL), 5% aqueous NaHCO₃ (3×70 mL), and dried over anhydrous Na₂ SO₄. Removal of the drying agent by filtration followed by in vacuo concentration of the filtrate afforded 4.30 g (84%) of an off-white crystalline solid, m.p. 100-112° C. Examination by ¹ H-NMR indicated methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1yl-2-iodo-L-phenylalaninate contaminated with approximately 4% of the (S,R)-diastereomer. The crude product was recrystallized twice from 1:1 EtOH--H₂ O to give 3.47 g (68%) of pure (S,S)-diastereomer as a white crystalline solid, m.p. 114-116° C.

EXAMPLE 22 Methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate

To a dry 100 mL 3-necked flask equipped with a thermometer, a magnetic stirrer, and a reflux condenser were added 1.50 g of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-2-iodo-L-phenylalaninate, 0.149 g of Pd(Ph₃ P)₄ (Aldrich), and 35 mL of 1:1 MeOH:THF under a nitrogen flush. The solution was deoxygenated by bubbling nitrogen through for 10 min and was then saturated with CO by bubbling CO through for 10 min. The solution changed color from yellow to bright orange during this period. The reaction vessel was put under CO balloon pressure by attaching a CO filled balloon to the condenser via a gas inlet adapter. The reaction mixture was heated to 60° C. with stirring. After 3 h, analysis by tlc (SiO₂, 1:1 hexane:EtOAc) indicated no remaining starting material, a new component at R_(f) =0.40, and two minor components at R_(f) =0.90-0.95. The solution was cooled to RT and concentrated in vacuo to give a yellow solid. This material was purified by flash chromatography on 85 g of silica gel (1:1 hexane:EtOAc) to afford 1.23 g (93%) of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate as a white crystalline solid, m.p. 128-130° C.

EXAMPLE 23 Methyl 5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate

According to example 10, 1.10 g of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate was hydrogenolyzed in the presence of 0.214 g of 10% Pd(C) and 0.18 mL of acetyl chloride to afford 0.86 g (97%) of methyl-5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate HCl as a light yellow foam, m.p. 77-80° C.

EXAMPLE 24 Methyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin-9-yl)methyl)-amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-2-(methoxycarbonyl)-L-phenylalaninate

According to example 11, 0.50 g of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid was coupled with 0.55 g of methyl 5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate HCl using 0.17 mL of isobutyl chloroformate and 0.14 mL (2x) of N-methylmorpholine. The crude product was purified by flash chromatography on 250 g of silica gel (97:3→95.5 CH₂ Cl₂ :MeOH) to afford 0.29 g (30%) of methyl

N-(N-(4-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin -9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-2-(methoxycarbonyl)-L-phenylalaninate as a white powder, m.p. 180° C. (dec).

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.52 (s,1H, benzaquinazoline NH), 9.86 (s, 1H, aromatic CH), 8.52 (d, 1H, J=7.6, amide NH), 8.23 (d, 1H, J=8.8, aromatic CH), 8.03 (d, 1H, J=8.4, aromatic CH), 7.80 (d, 1H, J=7.2, aromatic CH), 7.59 (m, 2H, aromatic CH, 4-amino-benzoyl NH), 7.50 (t, 1H, J=7.6, aromatic CH), 7.44-7.21 (m, 5H, aromatic CH, amide NH), 6.53 (d, 1H, J=8.0, aromatic CH), 6.40 (d, 1H, J=14.5, aromatic CH), 4.58 (m, 3H, benzoquinazoline 9-CH₂, methine), 4.45 (m, 1H, methine), 3.82(s, 3H, ArCO₂ CH₃), 3.57 (s, 3H, CO₂ CH₃), 3.55 (s, 3H, CO₂ CH₃), 3.48 (m, 1H, phe Ar--CH₂), 3.10 (m, 1H, phe Ar--CH₂), 2.45 (s, 3H, CH₃), 2.27 (t, 2H, J=7.5, glu 4-CH₂), 2.01-1.72 (m, 2H, glu 3-CH₂).

EXAMPLE 25 N-(N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl)-2-carboxy-L-phenylalanine

A 50 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 0.11 g of methyl

N-(N-(4-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-2-(methoxycarbonyl)-L-phenylalaninate and 15 mL of 1:1 THF:H₂ O. The solution was treated with 62 mg of LiOH (Fisher Scientific Co., Fair Lawn, N.J.) and stirred at RT under nitrogen. After 18 h, analysis by HPLC (C18, 70:30:0.1 H₂ O:MeCN:TFA) showed no remaining starting material (k'=4.89), a new component at K'=2.29 (92%), and three minor components between k'=0.72 and 1.13. The solution was concentrated in vacuo to 7 mL and subjected to semipreparative HPLC (C18, 80:20:0.1.ae butted.70:30:0.1 H₂ O:MeCN:TFA over 25 min). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo to dryness. The residue was dissolved in 20 mL of 0.17 M aqueous LiOH, filtered, and acidified to pH=3.5 by addition of 1 N aqueous HCl. A white solid resulted which was separated by centrifugation and washed with four cycles of aqueous suspension-centrifugation-decantation. The product was suspended in 10 mL of water, frozen, and lyophilized to afford 60 mg (56%) of N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl)-2-carboxy-L-phenylalanine as a fluffy white solid, m.p. 245° C. (dec).

HPLC: one peak on C18, K'=1.86, 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.00-11.70 (br s, COOH), 12.54 (s, 1H, benzoquinazopline NH), 9.84 (s, 1H, aromatic CH), 8.35 (d, 1H, J=7.9, amide NH), 8.22 (d, 1H, J=9.1, aromatic CH), 8.01 (d, 1H, J=8.2, aromatic CH), 7.78 (d, 1H, J=7.8, aromatic CH), 7.60 (m, 2H, aromatic CH, 4-aminobenzoyl NH), 7.50 (t, 1H, J=7.8, aromatic CH), 7.45-7.18 (m, 5H, aromatic CH, amide NH), 6.53 (d, 1H, J=8.5, aromatic CH), 6.39 (d, 1H, J=14.7, aromatic CH), 4.67-4.46 (m, 3H, benzoquinazoline 9-CH₂, methine), 4.41 (m, 1H, methine), 3.55 (dd; 1H; J=4.0, 12.3; phe Ar--CH₂), 3.06 (dd, 1H, J=10.2, 12.9; phe Ar--CH₂), 2.42 (s, 3H, CH₃), 2.16 (t, 2H, J=7.3, glu 4-CH₂), 1.98-1.64 (m, 2H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₆ H₃₂ N₅ O₉ F.H₂ O (MW 715.69): C, 60.42; H, 4.79; N, 9.79. Found C, 60.49; H, 4.83; N, 9.76.

Mass Spectrum: (Ion Spray) 698 (M+H)⁺, 489, 361.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 265.3 (43700), 298.4 (25600), 347.9 (5320). 1_(min) (e) 241.5 (20300), 284.9 (24600), 340.0 (3140).

EXAMPLE 26 Methyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate

According to example 13, 0.29 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was reacted with 0.32 g of methyl 5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate HCl using 0.35 mL of DECP (Aldrich) and 0.58 mL of Et₃ N. The crude product was purified by flash chromatography on 100 g of silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.27 (51%) of methyl

N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl-L-glutam-1-yl-2-(methoxycarbonyl)-L-phenylalaninate as a yellow powder, m.p. 125-130° C.

EXAMPLE 27 N-(N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)-2-carboxy-L-phenylalanine

To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.12 g of methyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl-L-glutam-1-yl)-2-(methoxycarbonyl)-L-phenylalaninate and 10 mL of 1:1 H₂ O:THF. The mixture was treated with 29 mg of LiOH.H₂ O (J. T. Baker Chemical Co., Phillipsburg, N.J.) and stirred at RT under nitrogen. After 48 h, analysis by HPLC (C18, 75:25:0.1 H₂ O:MeCN:TFA) indicated a major new component at k'=1.70 (ca. 90%) and three minor components at K'=0.60, 1.03, and 2.58. The solution was neutralized by dropwise addition of 1 N aqueous HCl and concentrated in vacuo to dryness. The residue was dissolved in 10 mL of MeOH was removed by rotary evaporation and the remaining suspension was frozen and lyophilized to afford 65 mg (45%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)-2-carboxy-L-phenylalanine as a yellow powder, m.p. 160° C. (dec).

HPLC: two peaks on C18, K'=2.49 (98%), k'=5.65 (2%); 78.22:0.1 H₂ O:MeCN:TFA; flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.10-11.90 (br5, COOH), 8.64 (s, 1H, pteridinyl 7-CH), 8.62 (br s, 1H, NH₂), 8.18 (d, 1H, J=8.0, amide NH), 7.92 (d, 1H, J=8.0, amide NH), 7.80-7.20 (br m, 2H, NH₂), 7.77 (d, 1H, J=7.3, aromatic CH), 7.70 (d, 2H, J=8.9, aromatic CH), 7.25 (m, 3H, aromatic CH), 6.81 (d, 2H, J=9.0, aromatic CH), 4.84 (s, 2H, pteridinyl-CH₂), 4.49 (m, 1H, methine), 4.39 (m, 1H, methine), 3.51 (m, 1H, phe Ar--CH₂), 3.24 (s, 3H, N--CH₃), 3.08 (m, 1H, phe AR--CH₂), 2.19 (t, 2H, J=7.6, glu 4-CH₂), 1.97-1.72 (m, 2H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₀ H₃₁ N₉ O₈.2.7H₂ O.1.2TFA (MW 831.10): C, 46.82; H, 4.56; N, 15.17. Found C, 46.76; H, 4.48; N, 15.15.

Mass Spectrum: (Ion Spray) 646 (M+H)⁺, 410, 369, 185.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 258.8 (23400), 307.0 (24200), 372.8 (7540). 1_(min) (e) 242.0 (16000), 273.2 (16200), 344.3 (5900).

EXAMPLE 28 Dimethyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-L-aspartate

According to example 9, 2.01 g of L-aspartic acid dimethylester HCl (Sigma) was coupled with 3.00 g of N-((benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid using 2.05 g of EDC (Aldrich) and 1.12 mL of N-methylmorpholine. This gave 3.03 g (88%) of dimethyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-L-aspartate as a crystalline white solid, m.p. 86-88° C.

EXAMPLE 29 Dimethyl 5-O-methyl-L-glutam-1-yl-L-aspartate

According to example 10, 2.00 g of dimethyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-L-aspartate was hydrogenolyzed in the presence of 0.45 g of 10% Pd(C) and 0.39 mL of acetyl chloride to afford 1.56 g (100%) of dimethyl 5-O-methyl-L-glutam-1-yl-L-aspartate HCl as a light yellow foam.

¹ NMR: (200 MHz, DMSO-d₆) δ 9.14 (d, 1H, J=7.4, NH), 8.40 (br s, 3H, NH₃ ⁺), 4.72 (m, 1H, glu methine), 3.89 (m, 1H, asp methine), 3.66 (s, 3H, CO₂ CH₃), 3.65 (s, 3H, CO₂ CH₃), 3.64 (s, 3H, CO₂ CH₃), 2.85 (d, 2H, J=6.2, asp CH₂), 2.49 (m, 2H, glu 4-CH₂), 2.04 (m, 2H, glu 3-CH₂).

EXAMPLE 30 Dimethyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-L-aspartate

According to example 11, 0.50 g of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9yl)methyl)amino)-2-fluorobenzoic acid was coupled with 0.45 g of dimethyl 5-O-methyl-L-glutam-1-yl-L-aspartate HCl using 0.17 mL of isobutyl chloroformate and 0.14 mL (2x) of N-methyl morpholine. The crude product was purified by flash chromatography on 250 g of silica gel (95:5 CH₂ Cl₂ :MeOH) to afford 0.33 g (38%) of dimethyl

N-(N-(4-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-L-aspartate as a white powder, m.p. 198-200° C.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 12.54 (s, 1H, benzoquinazoline NH), 9.85 (s, 1H, aromatic CH), 8.55 (d, 1H, J=7.7, amide NH), 8.23 (d, 1H, J=8.8, aromatic CH), 8.02 (d, 1H, J=8.4, aromatic CH), 7.70-7.45 (m, 4H, aromatic CH, amide NH), 7.33 (m, 1H, 4-amino-benzoyl NH), 6.55 (dd; 1H; J=8.8, 1.9; aromatic CH), 6.42 (dd; 1H; J=15.3, 1.7; aromatic CH), 4.72-4.41 (m, 4H, benzoquinazoline 9-CH₂, glu and asp methines), 3.62 (s, 3H, CO₂ CH₃), 3.60 (s, 3H, CO₂ CH₃), 3.55 (s, 3H, CO₂ CH₃), 2.91-2.66 (m, 2H, asp CH₂), 2.42 (s, 3H, CH₃), 2.34 (t, 2H, J=7.7, glu 4-CH₂), 2.13-1.80 (m, 2H, glu 3-CH₂).

EXAMPLE 31 N-(N-(4-((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-L-aspartic acid

According to example 12, 0.12 g of dimethyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-L-aspartic was saponified in 8 mL of 1:1 THF-H₂ O using 45 mg of LiOH.H₂ O (Baker). Analysis of the crude product by reverse phase HPLC (C18, 80:20:0.1 H₂ O:MeCN:TFA) indicated a major component at K'=1.73 (90%) and a minor component at K'=1.28. The material was purified by semi-preparative HPLC using gradient elution (C18, 80:20:0.1→75:25:0.1 H₂ O:MeCN:TFA over 20 min). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo. The residue was mixed with 20 mL of water and treated with enough LiOH.H₂ O to dissolve the solid. The solution was filtered and acidified to pH=3.0 by addition of 1 N HCl. A white solid precipitated which was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 57 mg (48%) of N-(N-(4-((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-L-aspartic acid as an off-white solid, m.p. 185° C. (dec). HPLC: one peak on C18, K'=1.42, 75:25:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.85-12.00 (br s, COOH), 12.53 (s, 1H, benzoquinazoline NH), 9.84 (s, 1H, aromatic CH), 8.36 (d, 1H, J=7.9, amide NH), 8.21 (d, 1H, J=8.8, aromatic CH), 8.01 (d, 1H, J=8.3, aromatic CH), 7.67-7.46 (m, 4H, aromatic CH, amide NH), 7.32 (m, 1H, 4-aminobenzoyl NH), 6.53 (d, 1H, J=8.5, aromatic CH), 6.40 (d, 1H, J=14.8, aromatic CH), 4.64-4.44 (m, 4H, benzoquinazoline 9-CH₂, glu methine, asp methine), 2.75-2.53 (m, 2H, asp CH₂), 2.42 (s, 3H, CH₃), 2.25 (t, 2H, J=7.3, glu 4-CH₂), 1.98 (m, 1H, glu 3-CH₂), 1.83 (m, 1H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₀ H₂₈ N₅ O₉ F.1.8H₂ O (MW 654.01): C, 55.10; H, 4.87; N, 10.71. Found: C, 55.04; H, 4.82; N, 10.61.

Mass Spectrum: (Ion Spray) 622 (M+H)⁺, 316, 213, 156.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 264.8 (46200), 298.4 (28500), 347.8 (6180).

1_(min) (e) 237.9 (19500), 285.7

(27900), 340.0 (3820).

EXAMPLE 32 Dimethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl-L-glutam-1-yl)-L-aspartate

According to example 13, 0.200 g of 4-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.18 g of dimethyl 5-O-methyl-L-glutam-1-yl-L-aspartate HCl using 0.24 mL of DECP (Aldrich) and 0.41 mL of Et₃ N. The crude product was purified by flash chromatography on 100 g of silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.17 g (53%) of dimethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl-L-glutam-1-yl)-L-aspartate as a yellow powder, m.p. 130-135° C.

EXAMPLE 33 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-aspartic acid (Procedure A)

For Procedure B to prepare this compound, see Example 125.

A 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.16 g of dimethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl-L-glutam-1-yl)-L-aspartate and 10 mL of 1:1 THF:H₂ O. The solution was treated with 44 mg of LiOH.H₂ O (Baker) and stirred at RT under N₂. After 3 h, tlc (SiO₂, 7:7:1 CH₂ Cl₂ : acetone:MeOH) indicated no remaining starting material at R_(f) =0.30 and a single spot at R_(f) =0.0. Analysis by reverse phase HPLC (C18, 85:15:0.1 H₂ O:MeCN:TFA) indicated a major component at k'=2.65 (90%) and a minor component at k'=1.94. The solution was neutralized by addition of 1 N HCl and concentrated in vacuo to dryness. The crude product was purified by semi-preparative HPLC (C18, 87:13:0.1 H₂ O:MeCN:TFA). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo to dryness. The residue was dissolved in 20 mL of H₂ O and lyophilized to afford 65 mg (32%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-aspartic acid as a yellow-orange powder, m.p. 165-180° C. (dec).

HPLC: one peak on C18, k'=2.45, 85:15:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.00-11.85 (br s, COOH), 9.17 (br, s, 1H, NH₂), 8.96 (br, s, 1H, NH₂), 8.71 (s, 1H, pteridinyl 7-CH), 8.60-8.30 (br, s, 1H, NH₂), 8.21 (d, 1H, J=8.0, amide NH), 8.06 (d, 1H, J=7.8, amide NH), 7.85-7.43 (br s, 1H, NH₂), 7.75 (d, 2H, J=8.7, aromatic CH), 6.82 (d, 2H, J=8.8, aromatic CH), 4.87 (s, 2H, pteridinyl-CH₂), 4.59-4.38 (m, 2H, 2 methines), 3.25 (s, 3H, N--CH₃), 2.75-2.53 (m, 2H, asp CH₂), 2.28 (t, 2H, J=7.5, glu 4-CH₂), 2.01 (m, 1H, glu 3-CH₂), 1.88 (m, 1H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₂₄ H₂₇ N₉ O₈.1.9H₂ O.1.6TFA (MW 786.20): C, 41.55; H, 4.15; N, 16.03. Found: C, 41.53; H, 4.19; N, 16.00

Mass Spectrum: (Ion Spray) 570 (M+H)⁺, 223.

UV Spectrum: (pH7 Buffer)λ_(max) (ε) 258.7 (24400), 307.3 (26800), 372.9 (8080). 1_(min) (e) 240.0 (14800), 272.9 (17000), 345.0 (6420).

EXAMPLE 34 Diethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-L-glutamate

According to example 9, 3.17 g of L-glutamic acid diethyl ester HCl (Sigma) was coupled with 4.09 g of N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutamic acid using 2.66 g of EDC (Aldrich) and 1.45 mL of N-methylmorpholine. This gave 4.80 g (74%) of diethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-L-glutamate as a white crystalline solid, m.p. 104° C.

EXAMPLE 35 Diethyl 5-O-ethyl-L-glutam-1-yl-L-glutamate

To a 500 mL Parr bottle were added 0.75 of diethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-L-glutamate, 0.14 g of 10% Pd(C) and 60 mL of EtOH under a N₂ flush. The mixture was deoxygenated by bubbling N₂ through for 7 min, treated with 1.52 mL of 1 M ethereal HCl, and hydrogenated at 45 psi for 18 h. The bottle was purged with N₂, catalyst was removed by filtration through celite, and the filtrate was concentrated in vacuo to dryness to afford 0.60 (99%) of diethyl 5-O-ethyl-L-glutam-1-yl-L-glutamate HCl as a light yellow foam. ¹ H-NMR: (300 MHz, DMSO-d₆) δ9.11 (d, 1H, J=7.5, NH), 8.41 (br s, 3H, NH₃ ⁺), 4.32 (m, 1H, methine), 4.06 (m, 6H, ethyl CH₂), 3.92 (m, 1H, methine), 2.47 (m, 4H, glu 4-CH₂), 2.12-1.79 (m, 4H, glu 3-CH₂), 1.19 (t, 9H, J=7.1, ethyl CH₃).

EXAMPLE 36 Diethyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazoling-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam-1-yl)-L-glutamate

According to example 11, 0.57 g of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazoling-9yl)methyl)amino)-2-fluorobenzoic acid was coupled with 0.60 g of diethyl 5-O-ethyl-L-glutam-1-yl-L-glutamate HCl using 0.20 mL of isobutyl chloroformate and 0.17 mL (2×) of N-methylmorpholine. The crude product was purified by flash chromatography on 350 g of silica get (EtOAc→98:2 EtOAc:MeOH) to afford 0.27 g (25%) of diethyl N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazoling-9-yl)methyl)amino)-2fluorobenzoyl)-5-O-ethyl-L-glutam-1yl)-L-glutamate as a white powder, m.p. 185-187° C.

EXAMPLE 37 N-(N-(4-(((1,2-Dihydro-3methyl-1-oxobenzo(f)quinazoline-9-yl)methyl)amino)-2fluorobenzoyl)-L-glutam-1-yl)-L-glutamic acid

A 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.217 g of diethyl N-(N-(4-(((1,2-dihydro-3methyl-1-oxobenzo(f)quinazoling-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam-1-yl)-L-glutamate, 40 mL of 0.2 N aqueous NaOH, and 15 mL of EtOH. The mixture was stirred at RT under N₂. The solid starting material slowly dissolved to give a clear solution. After 2 h, tlc (SiO₂, EtOAc) indicated no remaining starting material at R_(f) =0.15 and a new spot at R_(f) =0.0. The solution was acidified to pH=3.00 by addition of 1 N HCl. A white solid precipitated which was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.17 g (84%) of N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1yl)-L-glutamic acid as a white powder, m.p. 180° C. (dec). The product was determined to be pure by HPLC. HPLC: one peak on C18, K'=1.66, 75:25:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆) δ12.65-11.90 (br s, COOH), 12.53 (s, 1 H, benzoquinazoline NH), 9.84 (s, 1 H, aromatic CH), 8.29 (d, 1 H, J=7.5, amide NH), 8.22 (d, 1 H, J=8.9, aromatic CH), 8.01 (d, 1 H, J=8.0, aromatic CH), 7.60 (m, 3 H, aromatic CH, amide NH), 7.51 (t, 1 H, J=9.0, aromatic CH), 7.33 (m, 1 H, 4-aminobenzoyl NH), 6.54 (dd; 1 H; J=8.5, 1.9; aromatic CH), 6.41 (dd; 1 H; J=13.9, 1.4; aromatic CH), 4.57 (d, 2 H, J=5.2, benzoquinazoline 9CH₂), 4.49 (m, 1 H, methine), 4.22 (m, 1 H, methine), 2.43 (s, 3 H, CH₃), 2.28 (m, 4 H, glu 4-CH₂), 2.10-1.70 (m, 4 H, glu 3-CH₂). Elemental Analysis: Calcd. for C₃₁ H₃₀ N₅ O₉ F.2H₂ O (MW 671.64): C, 55.44; H, 5.10; N, 10.43. Found: C, 55.38; H, 5.12; N, 10.26.

Mass Spectrum: (FAB) 636 (M+H)⁺, 613, 581, 549.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 264.9 (45100), 296.1 (28000), 331.4 (7280), 348.1 (5990). ¹ min.sup.(e) 285.6 (27200), 329.3 (7040), 339.8 (3700). ¹ sh.sup.(e) 271.5 (41900).

EXAMPLE 38 Diethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-L-glutamate

According to example 13, 0.142 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.15 g of diethyl 5-O-ethyl-L-glutam-1-yl-L-glutamate-HCl using 0.18 mL of DECP (Aldrich) and 0.31 mL of Et₃ N. The crude product was purified by flash chromatography on 120 g of silica gel (7:71 CH₂ Cl₂ :acetone:MeOH) to afford 0.14 g (56%) of diethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-L-glutamate as a yellow powder, m.p. 105-109° C.

EXAMPLE 39 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamic acid (Procedure A)

For Procedure B to prepare this compound, see Example 129. According to example 33, 0.125 g of diethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1yl-L-glutamate was saponified in 10 mL of 1:1 THF:H₂ O using 23 mg of LiOH (Fisher). After stirring at RT under N₂ for 18 h, the solution was acidified to pH=2.0 by addition of 1 N HCl and was then concentrated in vacuo to dryness. Analysis of the residue by HPLC (C18, 85:15:0.1 H₂ O:MeCN:TFA) indicated a major component at K'=2.89 (90%) and two minor components at k'=2.46 and 1.17. The crude product was purified by semi-preparative HPLC (C18, 87:13:0.5 H₂ O:MeCN:TFA) and lyophilized to afford 37 mg (24%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamic acid as a yellow powder, m.p. 178° C.

HPLC: one peak on C18, K'=2.90, 85:15:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.20-(br s, COOH), 9.30 (br s, 1 H, NH₂), 9.10 (br s, 1 H, NH₂), 8.74 (s, 1 H, pteridinyl 7-CH), 8.72-8.42 (br s, 1 H, NH₂), 8.20 (d, 1 H, J=7.5, amide NH), 8.07 (d, 1H, J=7.5, amide NH), 7.76 (d, 2 H, J=32 8.8, aromatic CH), 7.61-7.40 (br s, 1 H, NH₂), 6.83 (d, 2 H, J=8.8, aromatic CH), 4.90 (s, 2 H, pteridinyl-CH₂), 4.43 (m, 1 H, methine), 4.20 (m, 1 H, methine), 3.27 (s, 3 H, N-CH₃), 2.30 (m, 4 H, glu 4-CH₂), 2.08-1.77 (m, 4 H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₂₅ H₂₉ N₉ O₈ :2.3H₂ O.1.7TFA (MW 818.84): C, 41.66; H, 4.35; N, 15.40. Found C, 41.61; H, 4.25; N, 15.44.

Mass Spectrum: (FAB) 584 (M+H)⁺, 309, 275, 176, 55, 119.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 220.9 (20500), 258.5 (21500), 306.7 (23200), 372.5 (7180). 1_(min) (e) 240.1 (13400), 272.5 (15100), 344.8 (5730).

EXAMPLE 40 3-Cyano-L-tyrosine

A solution of 5.70 g of 3-amino-L-tyrosine (Aldrich) in 35 mL of 2.15 N aqueous HCl was cooled to 0° C. and treated with a solution of 1.41 g of NaNO₂ (Aldrich) in 5 mL of water followed by 2.97 g of Na₂ CO₃. After stirring at 0° C. for 7 min, the solution was slowly added (via an addition funnel) to a stirred mixture of 3.58 g of CuCN (Aldrich), 5.88 g of NaCN (Aldrich) and 5.72 g of Na₂ CO₃ in 40 mL of water maintained at 75° C. in a 250 mL 3-necked flask equipped with a condenser. During the addition, vigorous evolution of nitrogen occurred and the reaction mixture changed color to dark red-orange. The reaction mixture was stirred at 75° C. for 2 h, heated to 95° C., and stirred for additional 1 h. After cooling to RT, the reaction mixture was acidified to pH=2.0 by addition of concentrated HCl via the addition funnel. The evolved gas was scrubbed through a 20% aqueous NaOH trap followed by a 5% NaOCl (Chlorox) trap . The mixture was filtered and the pH of the filtrate was adjusted to 6.0 with concentrated NH₄ OH. The brown solution was mixed with 300 mL of water and stirred with 300 g of DOWEX 50WX-8 ion exchange resin (acid form). The resin was filtered, washed with 1 L of water, mixed with 500 mL of water, and the mixture was basified to pH 11 by addition of concentrated NH₄ OH. The mixture was filtered and the filrate concentrated in vacuo to dryness. The dark brown residue was suspended in 20 mL of water and treated with enough 1 N aqueous NaOH to dissolve the solid. The pH was then adjusted to 6.0 by addition of 1 N aqueous HCl. A precipitate formed which was collected by filtration and dried in vacuo to afford 1.45 g (35%) of 3-cyano-L-tyrosine as a chocolate brown solid, m.p. >250° C. ¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.60-7.20 (br s. NH₂), 7.39 (s, 1 H, aromatic CH), 7.30 (m, 1 H, aromatic CH), 6.98 (m, 1 H, aromatic CH), 3.52 (m, 1 H, methine), 2.92 (m, 2 H, ArCH₂).

EXAMPLE 41 Methyl 3-(methoxycarbonyl)-L-tyrosinate

A 250 mL 3-necked flask equipped with a reflux condenser and a magnetic stirrer was charged with 1.45 g of 3-cyano-L-tyrosine, 50 mL of concentrated HCl, and 50 mL of water. The solution was heated to reflux with stirring. After 2 days, analysis of the reaction mixture by HPLC (C18, 90:10:0.1 H₂ O:MeCN:TFA) indicated a major component at k¹ =2.23 (75%) and a minor component at k¹ =1.59 (25%). After 4 days HPLC indicated only the k¹ =2.23 material. The reaction mixture was cooled to RT, filtered, and the filtrate was concentrated in vacuo to dryness to give 1.2 g of 3-carboxy-L-tyrosine as a dark brown solid. IR analysis (nujol) indicated loss of the nitrile stretch of 3-cyano-L-tyrosine, at 2223 cm⁻¹. The crude solid was added to 60 mL of anhydrous MeOH in a 250 mL 3-necked flask equipped with a condenser and a magnetic stirrer. The mixture was acidified by bubbling HCl gas through for 5 min and heated to reflux with stirring. After 6 h, the reaction mixture was cooled to RT and neutralized with solid NaHCO₃. The mixture was concentrated in vacuo to dryness, mixed with 100 mL of water, and extracted with CH₂ Cl₂ (4×50 mL). The combined extracts were washed with 5% aqueoous NaHCO₃ (2×50 mL), dried over anhydrous Na₂ SO₄, and concentrated in vacuo to 10 mL. The solution was diluted with 35 mL of ether and treated with 5.5 mL of 1 M etheral HCl. A solid precipitated which was collected by filtration, washed with ether, and dried in vacuo to afford 0.90 (50%) of methyl 3-(methoxycarbonyl)-L-tyrosinate.HCl as an off-white powder, m.p. 211-213° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 10.45 (s, 1 H, OH), 8.59 (br, s, 3 H, NH₃ ⁺), 7.66 (s, 1 H, aromatic CH), 7.38 (dd; 1 H: J=7.9, 1.1, aromatic CH), 6.97 (d, 1 H, J=7.9, aromatic CH), 4.26 (m, 1 H, methine), 3.89 (s, 3 H, CO₂ CH₃), 3.68 (s, 3 H, CO₂ CH₃), 3.09 (m, 2 H, ArCH₂).

EXAMPLE 42 Methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-tyrosinate

According to example 9, 0.08 g of methyl 3-(methoxycarbonyl)-L-tyrosinate.HCl was coupled with 0.82 g of N-(benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid using 0.56 g of EDC (Aldrich) and 0.30 mL of N-methylmorpholine. This gave 1.24 g (85%) of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-tyrosinate as a white solid, m.p. 142-144° C.

EXAMPLE 43 Methyl 5-O-methyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-tyrosinate

According to example 10, 0.99 g of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-tyrosinate was hydrogenolyzed using 0.19 g of 10% Pd(C) and 0.15 mL of acetyl chloride in 90 mL of 2:1 EtOH:EtOAc to afford 0.75 g (95%) of methyl 5-O-methyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-tyrosinate.HCl as a light yellow foam, m.p. 95-97° C. (dec).

EXAMPLE 44 Methyl N-(N-(4(((1,2-dihydro-3-methyl-1-oxobenzp(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-3-(methoxycarbonyl)-L-tyrosinate

According to example 11, 0.65 g of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazoling-9-yl)methyl)amino)-2)fluorobenzoic acid was coupled with 0.75 g of methyl 5-O-methyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-tyrosinate.HCl using 0.22 mL of isobutyl chloroformate and 0.19 mL (2×) of N-methylmorpholine. The crude product was purified by flash chromatography on 300 g of silica gel (99:1→94:6 EtOAc:MeOH) to afford 0.53 g (40%) of methyl N-(N-(4(((1,2-dihydro-3-methyl-1oxobenzo(f)quinazolin-9-methyl)amino)-2fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-3-(methoxycarbonyl)-L-tyrosinate as a white powder, m.p. 195-204° C. (dec).

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.52 (benzoquinazoline NH), 10.38 (s, 1 H, OH), 9.83 (s, 1 H, aromatic CH), 8.45 (d, 1 H, J=7.5, amide NH), 8.21 (d, 1 H, J=8.9, aromatic CH), 8.00 (d, 1 H, J=8.3, aromatic CH), 7.60 (m, 3 H, aromatic CH, 4-aminobenzoyl NH), 7.48 (m, 2 H, aromatic CH), 7.36 (m, 2 H, aromatic CH, amide NH), 6.86 (d, 1 H, J=8.6, aromatic CH), 6.52 (dd; 1 H; J=8.5, 1.9; aromatic CH), 6.39 (dd; 1 H; J=13.9, 1.5; aromatic CH), 4.57 (d 2 H, J=5.6, benzoquinazoline 9-CH₂), 4.45 (m, 2 H, methines), 3.84 (s, 3 H, CO₂ CH₃), 3.58 (s, 3 H, CO₂ CH₃), 3.52 (s, 3 H, CO₂ CH₃), 3.05-2.80 (m, 2 H, ArCH₂), 2.42 (s, 3 H, CH₃), 2.25 (t, 2 H, J=7.8, glu 4-CH₂), 2.00-1.77 (m, 2 H, glu 3-CH₂).

EXAMPLE 45 3-Carboxy-N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl)-L-tyrosine

A solution of 0.15 g of methyl N-(N-(4(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-1-yl)-3-(methoxycarbonyl)-L-tyrosinate in 20 mL of 0.2 N aqueous NaOH was stirred at RT under N₂ for 18 h. Analysis of the solution by HPLC (C18, 70:30:0.1 H₂ O:MeCN:TFA) indicated a major component at k'=1.9 (90%) and two minor components at k'=1.0 and 2.6. The solution was acidified to pH 2.5 by addition of 1 N HCl. A white precipitate resulted which was separated by centrifugation and purified by semi-preparative HPLC (C18, 75:25:0.1 H₂ O:MeCN:TFA). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo to dryness. The residue was dissolved in 20 mL of 0.15 N aqueous NaOH and the solution by HPLC (C18, 70:30:0.1 H₂ O:MeCN:TFA) indicated a major component at k'=1.9 (90%) and two minor component at k'=1.0 and 2.6. The solution was acidified to pH 2.5 by addition of 1 N HCl. A white precipitate resulted which was separated by centrifugation and purified by semi-preparative HPLC (C18, 75:25:0.1 H₂ O:MeCN:TFA). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo to dryness. The residue was dissolved in 20 mL of 0.15 N aqueous NaOH and the solution was acidified to pH=3.0 by addition of 1 N HCl. The resulting white precipitate was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 39 mg (26%) of 3-carboxy-N-(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl)-L-tyrosine as a white powder, m.p. 198-205° C. (dec).

HPLC: one peak on C18, k'=1.86, 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (200 MHz, DMSO-d₆) δ 13:00-11.00 (br m, COOH, benzoquinazoline NH), 9.86 (s, 1 H, aromatic CH), 8.28 (d, 1 H, J=7.5, amide NH), 8.24 (d, 1 H, J=8.9, aromatic CH), 8.03 (d, 1 H, J=8.4, aromatic CH), 7.70-7.42 (m, 5 H, aromatic CH, 4-aminobenzoyl NH), 7.40-7.24 (m, 2 H, aromatic CH, amide NH), 6.78 (d, 1 H, J=8.6, aromatic CH), 6.55 (dd; 1 H; J=8.5, 1.9: aromatic CH), 6.42 (dd; 1 H; J=13.9, 1.5; aromatic CH), 4.58 (d, 2 H, J=5.8, benzoquinazoline 9-CH₂), 4.55-4.31 (m, 2 H, methines), 3.06-2.75 (m, 2 H, ArCH₂), 2.24 (s, 3 H, CH₃), 2.21 (t, 2 H, J=7.6, glu 4-CH₂), 2.08-1.70 (m, 2 H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₆ H₃₂ N₅ O₁₀ F.2.2 H₂ O (MW 753.31): C, 57.40; H, 4.87; N, 9.30. Found: C, 57.37; H, 4.97; N, 9.28.

Mass Spectrum: (FAB) 714 (M+H)⁺, 613, 549, 461, 360, 309.

UV Spectrum: (pH7 Buffer) ¹ _(max) (e) 265.9 (44700), 300.2 (28200), 348.3 (5270). ¹ _(min) (e) 246.3 (23400), 285.2 (25500), 340.8 (3630).

EXAMPLE 46 tert-Butyl 3-(tert-butoxycarbonyl)-L-tyrosinate

A 250 mL 3-necked flask equipped with a magnetic stirrer and a condenser was charged with 4.1 g of 3-cyano-L-tyrosine and 100 mL of 6 N aqueous HCl. The mixture was heated to reflux with stirring. After 36 h, HPLC (C18, 90:10:0.1 H₂ O:MeCN:TFA) indicated no remaining starting material at k'=1.33 and a major new component at k'=1.69. The reaction mixture was cooled to RT, filtered to remove solids, and concentrated in vacuo to dryness. The residue was dissolved in 100 mL of 8:2 H₂ O:MeOH and the solution was passed through two C18 plugs (3.5×4.5 cm, EM separations LiChroprep RP-18) washing with 8:2 H₂ O:MeOH. The filtrate was concentrated in vacuo to dryness to afford 4.5 g of 3-carboxytyrosine as a brown solid. A portion of this material (1.30 g) was dissolved in 40 mL of 1,4-dioxane with 2 mL of concentrated H₂ SO₄ and the solution was added to 30 mL of isobutylene (condensed at -78° C.) in a 300 mL pressure bottle equipped with a magnetic stirrer. The vessel was stoppered, warmed to RT and stirred. After 48 h, the bottle was cooled to 0° C. in an ice water bath, opened, and the mixture was treated with 20 g of NaHCO₃. After allowing the isobutylene to evaporate, the mixture was suspended in 300 mL of water and extracted with CH₂ Cl₂ (4×100 mL). The combined CH₂ Cl₂ extracts were washed with 5% aqueous NaHCO₂ (2×50 mL), dried over anhydrous MgSO₄, and concentrated in vacuo to give a yellow-brown oil. Analysis by tlc (SiO₂, 97:3 CH₂ Cl₂ :MeOH) indicated a major component at R_(f) =0.41 and three minor components at R_(f) =0.30, 0.25 and 0.0. The crude material was purified by flash chromatography on 75 g of silica gel (94:6 CH₂ Cl₂ :MeOH) to give 1.2 g of a light yellow oil. The oil was dissolved in 40 mL of ether and treated with 3.9 mL of 1 M ethereal HCl. A precipitate formed which was collected by filtration and dried in vacuo to afford 1.14 g (53%) of tert-butyl 3-(tert-butoxycarbonyl)-L-tyrosinate.HCl as a fluffy white solid. An analytical sample (26 mg) was prepared by recrystallization from MeOH-ether, m.p. 205° C. (dec). ¹ H-NHR: (300 MHz, DMSO-d₆) δ 10.69 (s, 1 H, OH), 8.39 (br s, 3 H, NH₃ ⁺), 7.57 (d, 1H, J=2.2, aromatic CH), 7.43 (dd; 1 H; J=8.6, 2.2; aromatic CH), 6.96 (d, 1 H, J=8.4, aromatic CH), 4.12 (m, 1 H, methine), 3.11 (dd; 1 H ; J=14.3, 5.3; ArCH₂), 2.97 (dd; 1 H, J=14.1, 8.1; ArCH₂), 1.57 (s, 9 H, t-Bu), 1.36 (s, 9 H, t-Bu).

EXAMPLE 47 tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonyl)-L-tyrosinate

According to example 9, 0.323 g of tert-butyl 3-(tert-butoxycarbonyl)-L-tyrosinate.HCl was coupled with 0.292 g of N-benzyloxycarbonyl-L-glutamic acid γ-t-butyl ester (Sigma) using 0.174 g of EDC (Aldrich) and 0.095 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 85 g of silica gel (75:25 hexane:EtOAc) to afford 0.49 g (89%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonyl)-L-tyrosinate as a tacky white foam.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 10.67 (s, 1 H, OH), 8.21 (d, 1 H, J=7.4, NH), 7.56 (s, 1 H, aromatic CH), 7.46-7.26 (m, 7 H, aromatic CH), 5.00 (m, 2 H, PhCH₂ O), 4.31 (m, 1 H, methine), 4.06 (m, 1 H, methine), 2.89 (d, 2 H, J=6.9, ArCH₂), 2.21 (t, 2 H, J=7.7, glu 4-CH₂), 1.92-1.64 (m, 2 H, glu 3-CH₂), 1.57 (s, 9 H, t-Bu), 1.38 (s, 9 H, t-Bu), 1.33 (s, 9 H, t-Bu).

EXAMPLE 48 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonyl)-L-tyrosinate

To a 300 mL Parr bottle were added 0.076 g of 10% Pd(C) and 20 mL of MeOH under a nitrogen flush. To this was added a solution of 0.49 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonyl)-L-tyrosinate in 50 mL of MeOH. The mixture was deoxygenated by bubbling nitrogen through for 7 min and was hydrogenated at 50 psi for 3 h. The vessel was purged with nitrogen, catalyst removed by filtration through celite, and the filtrate concentrated in vacuo to give a thick oil.

This material was purified by flash chromatography on 70 g of silica gel (98:2 CH₂ Cl₂ :MeOH) to afford 0.31 g (82%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3- (tert-butoxycarbonyl)-L-tyrosinate as a thick transparent oil. A small sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 5 mg of the product with excess 1 N ethereal HCl and concentrating to dryness.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 10.68 (s, 1 H, OH), 8.99 (d, 1 H, J=7.1, NH), 8.33 (br s, 3 H, NH₃ ⁺), 7.60 (d, 1 H; J=2.0, aromatic CH), 7.47 (dd: 1 H; J=8.6, 2.2; aromatic CH), 6.92 (d, 1 H, J=8.4, aromatic CH), 4.38 (m, 1 H, methine), 3.87 (m, 1 H, methine), 2.92 (d, 2 H, J=7.1, ArCH₂), 2.34 (m, 2 H, glu 4-CH₂), 2.00 (m, 2 H, glu 3-CH₂), 1.59 (5, 9 H, t-Bu), 1.41 (s,9 H, t-Bu), 1.35 (s, 9 H, t-Bu).

EXAMPLE 49 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonyl)-L-tyrosinate

A dry 100 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 25 mL of anhydrous DMF, 0.26 mL of DECP (Aldrich) and 0.28 mL of Et₃ N. To this solution was added 0.217 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic)benzoic acid hemihydrochloride dihydrate (Aldrich). The solid starting material slowly dissolved to give a light yellow solution. After stirring for 3 h under N₂, the solution was treated with 0.29 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonyl)-L-tyrosinate in 5 mL of DMF. The solution was stirred for 18 h at RT and was then concentrated in vacuo to dryness. The residue was dissolved in 85 mL of CHCl₃. The resulting solution was washed with 1% aqueous NH₄ OH (3×60 mL), dried over anhydrous MgSO₄, and concentrated to give a thick, yellow-orange oil. This material was purified by flash chromatography on 150 g of silica gel (7:7:1 CH₂ Cl₂ :acetone: MeOH) to afford 0.245 g (52%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonly)-L-tyrosinate as a light yellow powder, m.p. 140-143° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 10.64 (s, 1 H, OH), 8.57 (s, 1 H, pteridinyl 7-CH), 8.15 (d, 1 H, J=7.4, amide NH), 7.99 (d, 1 H, J=7.9, amide NH), 7.71 (d, 2 H, J=8.8, aromatic CH), 7.67 (br s, 1 H, NH₂), 7.53 (d, 1 H, J=2.0, aromatic CH), 7.43 (br s, 1 H, NH₂), 7.37 (dd; 1 H; J=8.6, 2.1; aromatic CH), 6.81 (m, 3 H, aromatic CH), 6.59 (br s, 2 H, NH₂), 4.79 (s, 2 H, pteridinyl-CH₂), 4.43 (m, 1 H, methine), 4.32 (m, 1 H, methine), 3.21 (s, 3 H, N-CH₃), 2.90 (d, 2 H, J=7.1, ArCH₂), 2.24 (t, 2 H, J=32 7.9, glu 4-CH₂), 2.03-1.78 (m, 2 H, glu 3-CH₂), 1.54 (5, 9H, t-Bu), 1.36 (s, 9 H, t-Bu), 1.30 (s, 9 H, t-Bu).

EXAMPLE 50 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-carboxy-L-tyrosine

A 100 mL 3-necked flask equipped with a magnetic stirrer and anitrogen inlet was charged with 0.20 g of tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(tert-butoxycarbonyl)-L-tyrosinate and 20 mL of Ch₃ NO₂. The yellow solution was stirred at RT and acidified by bubbling gaseous HCl through for 10 min. Upon HCl treatment, the color rapidly changed from yellow to orange to lighter yellow and a solid began to precipitate. The mixture was stirred at RT under N₂ for 1.5 h and was then concentrated in vacuo to dryness. The resulting yellow solid was suspended in 15 mL of water and treated with sufficient 1 N aqueous NaOH to give complete solution. The solution was filtered and neutralized by addition of 1 N aqueous HCl. A yellow precipitate resulted which was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 140 mg (88%) of N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-carboxy-L-tyrosine as a yellow-orange powder, m.p. 195° C. (dec).

HPLC: two peaks on C18; k'=3.16 (98%), k'=6.07 (2%); 80:20:0.1 H₂ O:MeCN:TFA:flow rate =1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.15-11.80 (br s, COOH), 8.62 (s, 1 H, pteridinyl 7CH), 8.19 (br s, 1 H, NH₂) 8.00 (m, 3 H, amide NH (2), NH₂), 7.72 (d, 2 H, J=8.7, aromatic CH), 7.61 (d, 1 H, J=2.1, aromatic CH), 7.22 (m, 3 H, NH₂ (2), aromatic CH), 6.81 (d, 2 H, J=8.7, aromatic CH), 6.66 (d, 1 H, J=7.8, aromatic CH), 4.81(s, 2 H, pteridinyl-CH₂), 3.39 (m, 2 H, methines), 3.21 (s, 3 H, N-CH₃), 2.94 (m, 1 H, ArCH₂), 2.86 (m, 1 H, ArCH₂), 2.27 (t, 2 H, J=7.9, glu 4-CH₂), 2.05-1.72 (m, 2 H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₀ H₃₁ N₉ O₉.1.8H₂ O (MW 694.06): C, 51.92; H, 5.02; N, 18.16. Found: C, 51.84; H, 5.0Z; N, 18.19.

Mass Spectrum: (Ion Spray)662(M+H)⁺, 385, 306, 235, 157.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 258.6 (19900), 306.3 (23500), 372.6 (54900). 1_(min) (e) 245.4 (14800), 272.8 (12600), 347.0 (4040).

EXAMPLE 51 (2R, 3S, 5S)-Benzyl 3-(3-iodobenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate

A dry 500 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 3.0 g of benzyl (2R, 3S)-6oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich) and 180 mL of anhydrous THF. After gently warming the mixture to dissolve the solid starting material, the solution was cooled to -78° C. and was treated with 8.13 mL of 1 M sodium bis(trimethylsilyl)amide in THF (Aldrich) by slow addition via syringe through a rubber septum. The solution was stirred at -78° C. under N₂ for 40 min and was then treated with a solution of 2.41 g of 3-iodobenzyl bromide (Lancaster, Windham, NH 03087) in 5 mL of anhydrous THF. The reaction mixture was stirred at -78° C. for 2.5 h, mixed with 100 mL of water and the resulting mixture was concentrated in vacuo to dryness. The residue was dissolved in 100 mL of EtOAc, washed with water (3×100 mL), dried over anhydrous Na₂ SO₄, and concentrated in vacuo to dryness to give a tan solid. Analysis by tlc (SiO₂, 8:2 hexane:EtOAc) indicated no remaining starting material at R_(f) =0.35, a major new component at R_(f) =0.50 and some unreacted 3-iodobenzyl bromide at R_(f) =0.80. The crude product was purified by flash chromatography on 150 g of silica gel (8:2 hexane:EtOAc) to afford 2.8 g (60%) of (2R, 3S, 5S)-benzyl 3-(3-iodobenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate as a white crystalline solid, m.p. 167-168° C.

EXAMPLE 52 (2R, 3S, 5S)-Benzyl 3-(3-methoxycarbonyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate

A dry 100 mL 3-necked flask equipped with a magnetic stirrer, a reflux condenser, and a thermometer was charged with 2.56 g of (2R, 3S, 5S)-benzyl 3-(3-iodobenzyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate and 50 mL of 1:1 THF:MeOH. The mixture was deoxygenated by bubbling N₂ through for 10 min and was treated with 1.0 mL of Et₃ N followed by 0.24 g of Pd(Ph₃ P)₄ (Aldrich). The mixture was saturated with CO and was heated to reflux (65° C.) with stirring. A slight positive pressure of CO was maintained by attaching a CO gas inlet adapter equipped with a mineral oil bubbler to the condenser. After 4 h at reflux, tlc (SiO₂, 8:2 hexane:EtOAc) indicated no remaining starting material at R_(f) =0.50 and a new component at R_(f) =0.30. The reaction mixture was cooled to RT at which point a voluminous white solid precipitated. The suspension was mixed with 100 mL of CH₂ Cl₂ and the resulting solution was filtered through a silica gel plug (2.5×3 cm) and concentrated in vacuo to give a light yellow solid. This material was purified by flash chromatography on 150 g of silica gel (8:2 hexane:EtOAc) to afford 2.0 g (88%) of (2R, 3S, 5S)-benzyl 3-(3-methoxycarbonyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate as an off-white crystalline solid, m.p. 180-182° C.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.05-7.80 (m, 2 H, aromatic CH), 7.72-6.97 (m, 14 H, aromatic CH), 6.73 (m, 1 H, aromatic CH), 6,58 (m, 2 H, aromatic CH), 6.25, 6.16 (d, J=2.2; d, J=2.6; 1 H total; 2 resonances for morpholine 2-H due to carbamate conformers), 5.36, 5.31 (d, J=2.6; J=2.6; 1 H total; 2 resonances for morpholine 3-H due to carbamate conformers), 5.16-4.88 (m, 3 H, morpholine 5-H, PhCH₂ O), 3.88, 3.85 (5, 5, 3 H total, CO₂ CH₃, conformers), 3.71-3.40 (m, 2 H, ArCH₂).

EXAMPLE 53 3-Methoxycarbonyl-L-phenylalanine

To a 500 mL Parr bottle were added 2.0 g of (2R, 3S, 5S)-benzyl 3-(3-methoxycarbonyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate, 0.46 g of PdCl₂ (Aldrich) and 60 mL of 1:1 THF:MeOH. The mixture was deoxygenated by bubbling N₂ through for 10 min and was then hydrogenolyzed at 45 psi for 18 h. The bottle was purged with N₂ and the mixture filtered to remove catalyst. The filtrate was concentrated in vacuo to 5 mL and triturated with addition of 50 mL of ethr. A white solid precipitated which was collected by vacuum filtration, washed with 30 mL of ether, and dried in vacuo to afford 0.88 g (91%) of 3-methoxycarbonyl-L-phenylalanine.HCl, m.p. >200° C.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.50 (br s, 3 H, NH₃ ⁺), 7.89 (m, 2 H, aromatic CH), 7.67-7.43 (m, 2 H, aromatic CH), 4.21 (m, 1 H, methine), 3.87 (s, 3 H, CO₂ CH₃), 3.22 (d, 2 H, J=6.5, ArCH₂).

EXAMPLE 54 tert-Butyl 3-methoxycarbonyl-L-phenylalaninate

A solution of 0.88 g of 3-methoxycarbonyl-L-phenylalanine.HCl, and 1.5 mL of conc. H₂ SO₄ in 20 mL of 1,4-dioxane was added to 25 mL of liquid isobutylene (condensed at -78° C. ) in a 300 mL pressure bottle equipped with a magnetic stirrer. The bottle was sealed and the mixture allowed to warm to RT with stirring. The initially heterogeneous mixture afforded a slightly cloudy solution after stirring for 18 h at RT. After 48 h the vessel was chilled in an ice water bath, opened, and the contents treated with 13 g of said NaHCO₃. The isobutylene was allowed to evaporate and the remaining mixture was concentrated in vacuo to dryness. The residue was suspended in 100 mL of water and extracted with EtOAc (4×40 mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous MgSO₄, and concentrated in vacuo to give an oil. This material was dissolved in 30 mL of ether and the solution was treated with 3.4 mL of 1 M ethereal HCl. A white solid precipitated which was collected by filtration and dried in vacuo to afford 0.79 g (74%) of tert-butyl 3-methoxycarbonyl-L-phenylalaninate.HCl, m.p. 165° C. (dec).

EXAMPLE 55 tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-L-glutam-1-yl-3-methoxycarbonyl-L-phenylalaninate

According to example 9, 0.79 g of tert-butyl 3-methoxycarbonyl-L-phenylalaninate.HCl was coupled with 0.84 g of N-benzyloxycarbonyl-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.50 g of EDC (Aldrich) and 0.27 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 85 g of silica get (70:30 hexane:EtOAc) to afford 1.38 g (93%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-L-glutam-1-yl-3-methoxycarbonyl-L-phenylalaninate as a transparent glass.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.26 (d, 1 H, J=7.5, NH), 7.80 (m, 2 H, NH, aromatic CH), 7.43 (d, 1 H, J=7.7, aromatic CH), 7.44-7.23 (m, 7 H, aromaic CH), 4.99 (m, 2 H, PhCH₂ O), 4.38 (m, 1 H, methine), 4.01 (m, 1 H, methine), 3.83 (s, 3 H, CO₂ CH₃), 3.02 (m, 2 H, ArCH₂), 2.19 (t, 2 H, J=7.7, glu 4-CH₂), 1.90-1.61 (m, 2 H, glu 3-CH₂), 1.38 (s, 9 H, t-Bu), 1.32 (s, 9 H, t-Bu).

EXAMPLE 56 tert-Butyl 5-O-tert-L-glutam-1-yl-3-methoxycarbonyl-L-phenylalaninate

According to example 48, 1.38 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-L-glutam-1-yl-3-methoxycarbonyl-L-phenylalaninate was hydrogenolyzed using 0.23 g of 10% Pd(C) in 60 mL of MeOH. This afforded 1.0 g (94%) of tert-butyl 5-O-tert-L-glutam-1-yl-3-methoxycarbonyl-L-phenylalaninte as a thick, transparent oil. The product was carried on to the next step without purification. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 7 mg of the product with excess 1 M ethereal HCl and concentrating to dryness.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 9.12 (d, 1 H, J=7.6, NH), 8.39 (br s, 3 H, NH₃ ⁺), 7.82 (m, 2 H, aromatic CH), 7.60 (d,1 H, J=7.5, aromatic CH), 7.46 (t, 1 H, J=6.9, aromatic CH), 4.40 (m, 1 H, methine), 3.85 (m, 1 H, methine), 3.83 (s, 3 H, CO₂ CH₃), 3.05 (d, 2 H, J=8.4, ArCH₂), 2.33 (t, 2 H, J=7.7, glu 4-CH₂), 1.97 (m, 2 H, glu 3-CH₂), 1.37 (s, 9 H, t-Bu), 1.31 (s, 9 H, t-Bu).

EXAMPLE 57 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-phenylalaninate

According to example 49, 0.200 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.245 g of tert-butyl 5-O-tert-L-glutam-1-yl-3-methoxycarbonyl-L-phenylalaninate using 0.24 mL of DECP (Aldrich) and 0.26 mL of Et₃ N in 20 mL of DMF. The crude product was purified by flash chromatography on 130 g of silica gel (12:12:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.297 g (73%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-phenylalaninate as yellow powder, m.p. 130° C. (dec).

EXAMPLE 58 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-carboxy-L-phenylalanine

According to example 50, 0.15 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-phenylalaninate was treated with gaseous HCl in 20 mL of CH₃ NO₂. After stirring the mixture at RT for 1 h, the mixture was concentrated in vacuo to dryness to give a yellow-orange solid. Analysis of this material by ¹ H-NMR indicated complete loss of the tert-butyl groups of the starting material. HPLC (C18, 75:25:0.1 H₂ O:MeCN:TFA) indicated a single component at k'=3.50. The intermediate methyl ester was dissolved in 25 mL of 1:1 THF:H₂ O, treated with 49 mg of LiOH.H₂ O (Baker), and the solution was stirred at RT under N₂ for 18 h. Analysis by HPLC indicated n remaining k'=3.50 material and a single new component at k'=1.58. The solution was neutralized by addition of 1 N aqueous HCl. A yellow slurry resulted which was concentrated in vacuo to 2 mL and diluted with 20 mL of water. The precipitate was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.109 g (84%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-carboxy-L-phenylalanine as a yellow-orange powder, m.p. 195° C. (dec).

HPLC: one peak on C18, k'=1.93, 78:22:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.00-12.10 (br s, COOH), 8.58 (s, 1 H, pteridinyl 7-CH), 8.08 (d, 1 H, J=7.8, amide NH), 7.98 (d, 1 H, J=8.0, amide NH), 7.80 (s, 1 H, aromatic CH), 7.79-7.60 (m, 4 H, NH₂ (1 ), aromatic CH), 7.49 (br s, 1 H, NH₂), 7.44 (d, 1 H, J=7.7, aromatic CH), 7.30 (t, 1 H, J=7.6, aromatic CH), 6.81 (d, 2 H, J=9.0, aromatic CH), 6.63 (br s, 2 H, NH₂), 4.79 (s, 2 H, pteridinyl-CH₂), 4.41 (m, 2 H, methines), 3.20 (s, 3 H, N-CH₃), 3.10 (m, 1 H, ArCHz), 2.97 (m, 1 H, ArCH₂), 2.23 (t, 2 H, J=7.8, glu 4-CH₂), 2.00-1.73 (m, 2 H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₀ H₃₁ N₉ O₈.1.5H₂ O (MW 672.65) C, 53.57; H, 5.09; N, 18.74. Found: C, 53.58; H, 5.11; N, 18.72.

Mass Spectrum: (Ion Spray) 646 (M+H)⁺, 437, 340, 308, 175.

UV Spectrum: (pH7 Buffer)λ_(max) (ε) 258.7 (24300), 307.5 (25000), 372.7 (7880). λ_(min) (ε) 244.0 (17900), 272.7 (16900), 346.2 (6440).

EXAMPLE 59 tert-Butyl N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)-quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-phenylalaninante

A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.150 g of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid, 0.221 g of tert-butyl 5-O-tert-L-glutam-1-yl-3-methoxycarbonyl-L-phenylalaninate, 65 mg of 3-hydroxy-1,2,3-benzotriazin-4(3 H)-one (Aldrich), and 24 mL of anhydrous DMF. The resulting light yellow solution was treated with 84 mg of EDC (Aldrich) and was stirred at RT under N₂ for 48 h. The DMF was removed in vacuo and the residue purified by flash chromatography on 150 g of silica gel (98:2→96:4 CH₂ Cl₂ :MeOH) to afford 0.255 g (78%) of tert-butyl N-4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)-quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-phenylalaninate as a white powder, m.p. 155-157° C.

EXAMPLE 60 N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-3-carboxy-L-phenylalanine

According to example 50, 0.219 g of tert-butyl N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)-quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(methoxycarbonyl)-L-phenylalaninate was treated with gaseous HCl in 20 mL of CH₃ NO₂. After removal of the CH₃ NO₂ in vacuo, the residue was saponified with 66 mg of LiOH.H₂ O (Baker) in 20 mL of 1:1 THF:H₂ O for 18 h. The solution was acidified to pH=2.5 by addition of 1 N HCl and the resulting precipitate was isolated and lyophilized in the usual manner to afford 0.162 g (84%) of N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-3-carboxy-L-phenylalanine as a white powder, m.p. 187° C. (dec.)

HPLC: one peak on C18, k'=4.29, 75:25:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.00-12.25 (br m, COOH, benzoquinazoline NH), 9.83 (s, 1 H, aromatic CH), 8.32 (d, 1 H, J=7.4, amide NH), 8.22 (d, 1 H, J=9.0, aromatic CH), 8.00 (d, 1 H, J=8.5, aromatic CH), 7.81 (s, 1 H, aromatic CH), 7.75 (d, 1 H, J=8.0, aromatic CH), 7.60 (m, 2 H, aromatic CH), 7.49 (m, 3 H, aromatic CH, 4-aminobenzoyl NH), 7.35 (m, 2 H aromatic CH, amide NH), 6.52 (d, 1 H, J=8.8, aromatic CH), 6.39 (d, 1 H, J=15.2, aromatic CH), 4.57 (br s, 2 H, benzoquinazoline 9-CH₂), 4.45 (m, 2 H, methines), 3.12 (m, 1 H, ArCH₂), 2,96 (m, 1 H, ArCH₂), 2.42 (s, 3 H, CH₃), 2.19 (m, 2 H, glu-4-CH₂), 2.01-1.70 (m, 2 H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₆ H₃₂ N₅ O₉ F.1.7H₂ O (MW 728.30): C, 59.37; H, 4.90; N, 9.62. Found: C, 59.45; H, 4.93; N, 9.42.

Mass Spectrum: (Ion Spray) 698 (M+H)⁺.

UV Spectrum: (pH7 Buffer) λ_(max) (δ) 265.3 (45500), 296.5 (26100), 348.0 (5530). λ_(min) (δ) 244.4 (22600), 287.0 (25500), 340.1 (3540).

EXAMPLE 61 2-Cyclopentylbenzyl alcohol

A 300 mL Parr bomb was charged with 10.0 g of 2-iodobenzyl alcohol (Aldrich), 80 mL of toluene, 30 mL of cyclopentene (Aldrich), 2.60 g of tri-ortho-tolylphosphine (Aldrich), and 6.6 mL of Et₃ N. The mixture was deoxygenated by bubbling N₂ through for 10 min and was then treated with 0.96 g of Pd(OAc)₂ (Aldrich). The bomb was flushed with N₂, sealed, and heated to 100° C. for 18 h. The vessel was cooled to RT, purged with N₂ and opened. The dark red reaction mixture was concentrated in vacuo to give a red-brown syrup. This material was dissolved in 200 mL of CH₂ Cl₂, washed with water (4×100 mL), dried over anhydrous MgSO₄, and concentrated in vacuo to afford a brown oil. Analysis by tlc (SiO₂, 8:2 hexane:EtOAc) indicated two new major components at R_(f) =0.45 and 0.50, tri-o-tolyphosphine at R_(f) =0.90, and 4 minor components at R_(f) =0.60, 0.70, 0.85 and 0.0. The crude product was purified by flash chromatography (twice) on 200 g of silica gel (9:1 hexane:EtOAc) to afford 6:1 g (82%) of a mixture of the R_(f) =0.45 and 0.50 materials. This material was determined to be a mixture of double bond isomers by ¹ H-NMR. The product (6.0g ) was then hydrogenated at 40 psi in 40 mL of MeOH in the presence of 0.60 g of 5% Pt(C) (Aldrich) for 4 h. The catalyst was removed by filteration through celite and the filtrate was concentrated in vacuo to afford 5.47 g (74% from 2-iodobenzyl alcohol) of 2-cyclopentylbenzyl alcohol as a light yellow-brown liquid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.40-7.07 (m, 4 H, aromatic CH), 5.06 (t, 1 H, J=5.4, OH), 4.58 (d, 2 H, J=5.4, ArCH₂ O), 3.24 (m, 1 H, cyclopentyl methine), 1.97 (m, 2 H, cyclopentyl CH₂), 1.89-1.42 (m, 6 H, cyclopentyl CH₂).

EXAMPLE 62 2-Cyclopentylbenzyl bromide

According to example 17, 5.47 g of 2-cyclopentylbenzyl alcohol was treated with 1.03 mL of PBr₃ in 25 mL of anhydrous CH₂ Cl₂ to afford 6.9 g (93%) of 2-cyclopentylbenzyl bromide as a clear liquid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.40-7.24 (m, 3 H, aromatic CH), 7.14 (m, 1 H, aromatic CH), 4.78 (s, 2 H, ArCH₂ Br), 3.31 (m, 1 H, cyclopentyl methine), 2.05 (m, 2 H, cyclopentyl CH₂), 1.88-1.47 (m, 6 H, cyclopentyl CH₂).

EXAMPLE 63 (2R, 3S, 5S)-Benzyl 3-(3-cyclopentylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate

According to example 51, 1.47 g of benzyl (2R, 3S)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich) was alkylated with 1.00 g of 2-cyclopentylbenzyl bromide using 3.99 mL of 1 M sodium bis(trimethylsilyl)amide in THF (Aldrich). The crude product was purified by flash chromatography on 125 g of silica gel (8:2 hexane:EtOAc) to afford 1.07 g (52%) of (2R, 3S, 5S)-benzyl 3-(3-cyclopentylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate as a white solid, m.p. 161-163° C.

EXAMPLE 64 2-Cyclopentyl-L-phenylalanine

According to example 53, 1.0 g of (2R, 3S, 5S)-benzyl 3-(3-cyclopentylbenzyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate was hydrogenolyzed in 40 mL of 1:1 THF:MeOH using 0.23 g of PdCL₂ to afford 0.373 g (76%) of 2-cyclopentyl-L-phenylalanine.HCl as an off-white powder, m.p. 175° C. (dec).

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.49 (br s, 3 H, NH₃ ⁺), 7.38-7.07 (m, 4 H, aromatic CH), 3.92 (m, 1 H, methine), 3.18 (m, 1 H, cyclopentyl methine), 1.98 (m, 2 H, cyclopentyl CH₂), 1.90-1.41 (m, 6 H, cyclopentyl CH₂).

EXAMPLE 65 tert-Butyl 2-cyclopentyl-L-phenylalaninate

According to example 53, 1.0 g of (2R, 3S, 5S)-benzyl 3-(3-cyclopentylbenzyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate was hydrogenolyzed in 40 mL of 1:1 THF:MeOH using 0.23 g of PdCl₂ to afford 0.373 g (76%) of 2-cyclopentyl-L-phenylalanine.HCl as an off-white powder, m.p. 175° C. (dec.).

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.49 (br s, 3 H, NH₃ ⁺), 7.38-7.07 (m, 4 H, aromatic CH), 3.92 (m, 1 H, methine), 3.18 (m, 1 H, cyclopentyl methine), 1.98 (m, 2 H, cyclopentyl CH₂), 1.90-1.41 (m, 6 H, cyclopentyl CH₂).

EXAMPLE 66 tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-2-cyclopentyl-L-phenylalaninate

According to example 54, 0.23 g of 2-cyclopentyl-L-phenylalanine.HCl was treated with 25 mL of isobutylene in 20 mL of 1,4-dioxane in the presence of 0.13 mL of conc. H₂ SO₄. Work-up afforded a clear oil which was dissolved in 20 mL of ether. The solution was treated with 1 mL of 1 M ethereal HCl and concentrated in vacuo to dryness to afford 0.28 g (100%) of tert-butyl 2-cyclopentyl-L-phenylalaninate.HCl as a light yellow glass.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.59 (br s, 3 H, NH₃ ⁺), 7.28 (m, 2 H, aromatic CH), 7.10 (m, 2 H, aromatic CH), 3.91 (m, 1 H, methine), 3.30 (m, 1 H, ArCH₂), 3.18 (m, 1 H, cyclopentyl methine), 2.99 (dd; 1 H; J=10.1, 12.4; ArCH₂), 1.98 (m, 2 H, cyclopentyl CH₂), 1.89-1.46 (m, 6 H, cyclopentyl CH₂), 1.19 (s, 9 H, t-Btu).

EXAMPLE 66 tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate

According to example 9, 0.25 g of tert-butyl 2-cyclopentyl-L-phenylalaninate.HCl was coupled with 0.26 g of N-benzyloxycarbonyl-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.16 g of EDC (Aldrich) and 0.084 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 85 g of silica gel (8:2 hexane:EtOAc) to afford 0.355 g (76%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate as a white solid, m.p. 95-96° C.

EXAMPLE 67 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate

According to example 48, 0.355 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate was hydrogenolyzed using 60 mg of 10% Pd(C) in 45 mL of MeOH. This afforded 0.236 g (85%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate as a thick, transparent oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.23 (m, 1 H, NH), 7.34-7.00 (m, 4 H, aromatic CH), 4.37 (m, 1 H, methine), 3.36-2.85 (m, 5 H, ArCH₂, cyclopentyl methine, NH₂), 2.27-1.92 (m, 4 H, glu 4-CH₂, glu 3-CH₂), 1.90-1.46 (m, 8 H, cyclopentyl CH₂), 1.39 (s, 9 H, t-Btu), 1.32 (s, 9 H, t-Bu).

EXAMPLE 68 tert-Butyl N-(4(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate

According to example 49, 0.189 g of a 4-(N-(2,4-diamino-6-pteridinylmthyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.236 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate using 0.23 mL of DECP (Aldrich) and 0.24 mL of Et₃ N in 20 mL of DMF. The crude product was purified by flash chromatography on 85 g of silica gel (12:12:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.195 g (50%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate as a yellow powder, m.p. 130-140° C.

EXAMPLE 69 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino(benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-phenylalanine

According to example 50, 0.167 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5O-tert-butyl-L-glutam-1-yl-2-cyclopentyl-L-phenylalaninate was treated with gaseous HCl in 15 mL of CH₃ NO₂ for 1 h. The reaction mixture was concentrated in vacuo to dryness to give a yellow solid. This material was suspended in ether, collected by vacuum filtration, and dried in vacuo to afford 1.15 g (91%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-phenylalanine as a light yellow powder, m.p. 155° C. (dec).

HPLC: one peak on C18, k'=3.25, 65:35:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.25-12.95 (br s, COOH), 9.31 (s, 1 H, NH₂), 9.09 (s, 1 H, NH₂), 8.80-8.58 (br s, 1 N, NH₂), 8.72 (s, 1 H, pteridinyl 7-CH), 8.24 (d, 1 H, J=8.0, amide NH), 8.01 (d, 1 H, J=8.0, amide NH), 7.74 (m, 3 H, NH₂ (1), aromatic CH), 7.22 (d, 1 H, J=8.1, aromatic CH), 7.12 (m, 2 H, aromatic CH), 6.93 (t, 1 H, J=7.4, aromatic CH), 6.82 (d, 2 H, J=9.0, aromatic CH), 4.88 (s, 2 H, pteridinyl-CH₂), 4.45 (m, 1 H, methine), 4.34 (m, 1 H, methine), 3.26-3.10 (m, 2 H, ArCH₂, cyclopentyl methine), 2.88 (m, 1 H, ArCH₂), 2.23 (t, 2 H, J=7.6, glu 4-CH₂), 2.06-1.40 (m, 10 H, glu 3-CH₂, cyclopentyl CH₂).

Elemental Analysis: Calcd. for C₃₄ H₃₉ N₉ O₆.1.7 HCl.2.2 H₂ O (MW 771.36): C, 52.94: H, 5.89; N, 16.34Cl, 7.81. Found: C, 52.57; H, 5.74; N, 16.13; Cl, 7.52.

Mass Spectrum: (Ion Spray) 670 (M+H)⁺, 613, 459, 391, 309, 275.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 259,.5 (24000), 308.5 (24000), 373.0 (7450). λ_(min) (ε) 241.0 (14400), 274.2 (16200), 346.5 (5840).

EXAMPLE 70 3-Cyclopentylbenzyl alcohol

According to example 61, 10.0 g of 3-iodobenzyl alcohol (Aldrich) was treated with 30 mL of cyclopentene using 0.96 g of Pd(OAc)₂, 2.60 g of tri-ortho-tolylphosphine, and 6.6 mL of Et₃ N. The resulting olefin mixture was hydrogenated for 2.5 h using 0.62 g of 5% Pt(C) in MeOH to afford 5.77 g (76%) of 3-cyclopentylbenzyl alcohol as a light yellow liquid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.32-7.17 (m, 2 H, aromatic CHO, 5.14 (t, 1 H, J=5.5, OH), 4.48 (d, 2 H, J=5.6, ArCH₂ O), 2.96 (m, 1 H, cyclopentyl methine), 2.01 (m, 2 H, cyclopentyl CH₂), 1.88-1.42 (m, 6 H, cyclopentyl CH₂).

EXAMPLE 71 3-Cyclopentylbenzyl bromide

According to example 17, 5.77 g of 3-cyclopentylbenzyl alcohol was treated with 1.09 mL of PBr₃ in 25 mL of anhydrous CH₂ Cl₂ to afford 7.85 g (100%) of 3-cyclopentylbenzyl bromide as a light yellow liquid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ7.37-7.14 (m, 4 H, aromatic CH), 4.68 (s, 2 H, ArCH₂ Br), 2.96 (m, 1 H, cyclopentyl methine), 2.00 (m, 2 H, cyclopentyl CH₂), 1.84-1.42 (m, 6 H, cyclopentyl CH₂).

EXAMPLE 72 (2R, 3S, 5S)-Benzyl 3-(3-cyclopentylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate

A dry 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 1.47 g of benzyl (2R, 3S)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich), 1.00 g of 3-cyclopentylbenzyl bromide, and 80 mL of anhydrous THF. The mixture was gently heated with stirring to dissolve the solid starting material and the resulting solution was cooled to -78° C. The solution was treated with 4.0 mL of 1 M sodium bis(trimethylsilyl)amide in THF (Aldrich) by slow addition via syringe through a rubber septum. After stirring at -78° C. for 4 h, the solution was mixed with 80 mL of water and worked-up according to example 51. The crude product was purified by flash chromatography on 85 g of silica gel (85:15 hexane:EtOAc) to afford 1.44 g (70%) of (2R, 3S, 5S)-Benzyl 3-(3-cyclopentylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate as a white powder, m.p. 82-84° C.

EXAMPLE 73 3-Cyclopentyl-L-phenylalanine

According to example 53, 1.37 g of (2R, 3R, 5S)-benzyl 3-(3-cyclopentylbenzyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate was hydrogenolyzed in 80 mL of 1:1 THF:MeOH using 0.31 g of PdCl₂ to afford 0.63 g (93%) of 3-cyclopentyl-L-phenylalanine.HCl as a white powder, m.p. >200° C.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.39 (br s, 3 H, NH₃ ⁺), 7.31-7.12 (m, 3 H, aromatic CH), 7.08 (d, 1 H, J=7.3, aromatic CH), 4.17 (m, 1 H, methine), 3.11 (d, 2 H, J=6.1, ArCH₂), 2.96 (m, 1H, cyclopentyl methine), 1.99 (m, 2H, cyclopentyl CH₂), 1.88-1.45 (m, 6H, cyclopentyl CH₂).

EXAMPLE 74 tert-Butyl 3-cyclopentyl-L-phenylalaninate

According to example 54, 0.50 g of 3-cyclopentyl-L-phenylalanine•HCl was treated with 25 mL of isobutylene in 20 mL of 1,4-dioxane in the presence of 0.3 mL of conc. H₂ SO₄. Work-up followed by acidification of the free amine with ethereal HCl afforded 0.49 g (82%) of tert-butyl 3-cyclopentyl-L-phenylalaninate•HCl as a white powder, m.p. 154-156° C.

EXAMPLE 75 tert-Butyl N-(benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

According to example 9, 0.49 g of tert-butyl 3-cyclopentyl-L-phenylalaninate•HCl was coupled with 0.51 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.30 g of EDC (Aldrich) and 0.16 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 80 g of silica gel (8:2 hexane:EtOAc) to afford 0.75 g (82%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a thick transparent oil.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.22 (d, 1H, J=7.3, NH), 7.33 (m, 6H, aromatic CH), 7.20-6.97 (m, 3H, aromatic CH, NH), 5.01 (m, 2H, PhCH₂ O), 4.33 (m, 1H, methine), 4.05 (m, 1H, methine), 2.91 (m, 3H, ArCH₂, cyclopentyl methine), 2.20 (t, 2H, J=7.7, glu 4-CH₂), 1.97 (m, 2H, glu 3-CH₂), 1.88-1.43 (m, 8H, cyclopentyl CH₂), 1.38 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu).

EXAMPLE 76 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

According to example 48, 0.75 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate was hydrogenolyzed using 0.12 g of 10% Pd(C) in 60 mL of MeOH. This afforded 0.58 g (99%) of tert-butyl-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 10 mg of the product with excess 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.99 (d, 1H, J=7.0, NH). 8.32 (br s, 3H, NH₃ ⁺), 7.24-7.01 (m, 4H, aromatic CH), 4.38 (m, 1H, methine), 3.85 (m, 1H, methine), 2.93 (m, 3H, ArCH₂, cyclopentyl methine), 2.34 (t, 2H, J=7.7, glu 4-CH₂), 1.96 (m, 4H, glu 3-CH₂, cyclopentyl CH₂), 1.82-1.44 (m, 6H, cyclopentyl CH₂), 1.40 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu).

EXAMPLE 77 tert-Butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

According to example 49, 0.200 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.25 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate using 0.24 mL of DECP (Aldrich) and 0.26 mL of Et₃ N in 20 mL of DMF. The crude product was purified by flash chromatography on 150 g of silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.195 g (47%) of tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a yellow powder, m.p. 115-120° C.

EXAMPLE 78 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine

According to example 50, 0.18 g of tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate was treated with gaseous HCl in 20 mL of CH₃ NO₂ for 1.25 h. Work-up and isolation in the usual manner afforded 0.135 g (84%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine as a yellow-orange powder, m.p. 170° C. (dec).

HPLC: one peak on C18, k'-=2.86, 65:35:0.1 H₂ O:MeCN:TFA, flow rate =1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.90-11.85 (br m, COOH), 8.58 (s, 1H, pteridinyl 7-CH), 8.04 (d, 1H, J=7.7, amide NH) 8.00 (d, 1H, J=8.1, amide NH, 7.72 (br s, 1H, NH₂), 7.71 (d, 2H, J=8.8, aromatic CH), 6.69 (br s, 2N, NH₂), 4.79 (s, 2H, pteridinyl-CH₂), 4.40 (m, 2H, methines), 3.22 (s, 3H, N-CH₃), 3.01 (m, 1H, ArCH₂), 2.94-2,.75 (m, 2H, ArCH₂, cyclopentyl methine), 2.24 (t, 2H, J=7.7, glu 4-CH₂), 2.03-1.76 (m, 4H, glu 3-CH₂, cyclopentyl CH₂), 1.73-1.35 (m, 6H, cyclopentyl CH₂).

Elemental Analysis: Calcd. for C₃₄ H₃₉ N₉ O₆ •1.6 H₂ O (MW 698.56): C, 58.46; H, 6.09; N, 18.05. Found: C, 58.50; H, 6.08; N, 18.10.

Mass Spectrum: (FAB) 670 (M+H)⁺, 613, 437, 309, 275.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 259.7 (23300), 308.3 (23500), 371.7 (7030). λ_(min) (ε) 241.0 (13500), 274.8 (15300), 346.1 (5260).

EXAMPLE 79 2-Cyclohexylbenzyl alcohol

According to example 61, 10.0 g of 2-iodobenzyl alcohol was treated with 25 mL of cyclohexene (Aldrich) using 0.96 g of Pd(O Ac)₂, 2.60 g of tri-ortho-tolyphosphine, and 6.6 mL of Et₃ N at 100° C. for 48 h. The resulting olefin mixture was hydrogenated for 6.5 h using 0.36 g of 5% Pt(C) in MeOH to afford 3.37 g (42%) of 2-cyclohexyl benzyl alcohol as a light yellow liquid.

¹ H: (300 MHz, DMSO-d₆) δ 7.33 (d, 1H, J=7.4, aromatic CH), 7.27-7.08 (m, 3H, aromatic CH), 5.03 (t, 1H, J=5.4, OH), 4.34 (d, 2H, J=5.4, ArCH₂ O), 2.76 (m, 1H, cyclohexyl methine), 1.85-1.61 (m, 5H, cyclohexyl CH₂), 1.48-1.22 (m, 5H, cyclohexyl CH₂).

EXAMPLE 80 2-Cyclohexylbenzyl bromide

According to example 17, 3.36 g of 2-cyclohexylbenzyl alcohol was treated with 0.59 mL of PBr₃ in 25 mL of anhydrous CH₂ Cl₂ to afford 3.90 g of crude product (90% pure by ¹ H-NMR). This material was vacuum distilled via short path (1.0 mmHg, 117-120° C.) to afford 3.54 g (79%) of 2-cyclohexylbenzyl bromide as a clear liquid.

¹ H: (300 MHz, DMSO-d₆) δ 7.39 (d, 1H, J=5.7, aromatic CH), 7.29 (m, 2H, aromatic CH), 7.13 (m, 1H, aromatic CH), 4.76 (s, 2H, ArCH₂ Br), 2.86 (m, 1H, cyclohexyl methine), 1.83-1.63 (m, 5H, cyclohexyl CH₂), 1.53-1.20 (m, 5H, cyclohexyl (CH₂).

EXAMPLE 81 (2R, 3S, 5S)-tert-Butyl 3-(2-cyclohexylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate

To a dry 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was added a solution of 1.47 g of tert-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich) in 30 mL of THF and a solution of 1.00 g of 2-cyclohexylbenzyl bromide in 8 mL of THF. Both solutions had been dried over 3 Å molecular sieves for 24 h. Anhydrous THF was used for rinse (30 mL) to give a total solution volume of 68 mL. The resulting solution was cooled to -78° C. and was treated with 4.15 mL of 1M sodium bis-(trimethylsilyl) amide in THF (Aldrich) by slow addition via syringe through a rubber septum. After stirring at -78° C. for 3.5 h, the reaction mixture was mixed with 80 mL of water and subjected to rotary evaporation to remove THF. The resulting mixture was extracted with EtOAc (4×50 mL). The combined EtOAc extracts ere washed with saturated aqueous NaCl (2×70 mL), dried over anhydrous MgSO₄ and concentrated in vacuo to afford a white solid. Purification of this material by flash chromatography on 200 g of silica gel (8:2 hexane:EtOAc) afforded 1.2 g (58%) of (2R, 3S, 5S)-tert-butyl 3-(2-cyclohexylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate as a white, crystalline solid, m.p. 195-198° C.

EXAMPLE 82 (2R, 3S, 5S)-6-Oxo-2,3-diphenyl-5-(2-cyclohexylbenzyl)morpholine

A dry 50 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charges with 2.10 g of (2R, 3S, 5S)-tert-butyl 3-(2-cyclohexylbenzyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate and 33 mL of anhydrous CH₂ Cl₂. The solution was stirred at RT and treated with 3.3 mL of RFA. After 3.5 h, tlc (SiO₂, 85:15 hexane:EtOAc) indicated no remaining starting material, a major new component at R_(f) =0.45, and two minor components at R_(f) =0.68 and 0.70. The solution was neutralized by addition of 9 mL of Et₃ N and was concentrated in vacuo to give a thick oil. This material was dissolved in 80 mL of CH₂ Cl₂, washed with water (3×60 mL) followed by 5% aqueous NaHCO₃ (3×60 mL and dried over anhydrous MgSO₄. Drying agent was removed by filtration and the filtrate concentrated in vacuo to afford a white solid. Purification of the crude product by flash chromatography on 150 g of silica gel (8:2 hexane:EtOAc) followed by recrystallization from EtOH-H₂ O afforded 1.27 g (75%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(2-cyclohexylbenzyl)morpholine as a white crystalline solid, m.p. 159-160° C.

EXAMPLE 83 2-Cyclohexyl-L-phenylalanine

According to example 53, 1.17 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(2-cyclohexylbenzyl)morpholine was hydrogenolyzed in 60 mL of 1:1 THF:EtOH using 0.34 g of PdCl₂ to afford 0.83 g of 2-cyclohexyl-L-phenylalanine•HCl as a fluffy white solid, m.p. 140° C. (dec).

EXAMPLE 84 tert-Butyl 2-cyclohexyl-L-phenylalaninate

According to example 54, 0.73 g of 2-cyclohexyl-L-phenylalanine•HCl was treated with 25 mL of isobutylene in 25 mL of 1,4-dioxane in the presence of 0.4 mL of conc. H₂ SO₄. Work-up followed by acidification of the free amine with ethereal HCl afforded 0.70 g (80%) of tert-butyl 2-cyclohexyl-L-phenylalaninate•HCl as a yellow foam.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.53 (br s, 3H, NH₃ ⁺), 7.27 (m, 2H, aromatic CH), 7.10 (m, 2H, aromatic CH), 3.86 (m, 1H, methine), 3.24 (m, 1H, ArCH₂), 2.97 (m, 1H, ArCH₂), 2.72 (m, 1H, cyclohexyl methineJ), 1.86-1.60 (m, 5H, cyclohexyl CH₂), 1.56-1.30 (m, 5H, cyclohexyl CH₂), 1.19 (s, 9H, t-Bu).

EXAMPLE 85 tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenyalaninate

According to example 9, 0.70 g of tert-butyl-2-cyclohexyl-L-phenylalaninate•HCl was coupled with 0.70 g of N-Cbz-L-glutamic acid-γ-tert-butyl ester (Sigma) using 0.42 g of EDC (Aldrich) and 0.23 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 150 g of silica gel (8:2 hexane:EtOAc) to afford 0.97 g (76%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenyalaninate as a white foam.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.32 (d, 1H, J=7.6, NH), 7.40-6.97 (m, 10H, aromatic CH, NH), 5.00 (m, 2H, PhCH₂ O), 4.26 (m, 1H, methine), 4.04 (m, 1H, methine), 3.07 (m, 1H, ArCH₂), 2.93-2.70 (m, 2H, ArCH₂, cyclohexyl methine, 2.19 (t, 2H, J=7.7, glu 4-CH₂), 1.90-1.62 (m, 7H, glu 3-CH₂, cyclohexyl CH₂), 1.56-1.33 (m, 5H, cyclohexyl CH₂), 1.38 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu).

Mass Spectrum: (CI, CH₄) 623 (M+H)⁺, 567, 539, 511, 489, 433, 193.

EXAMPLE 86 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenyalaninate

According to example 48, 0.97 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenyalaninate was hydrogenolyzed using 0.16 g of 10% Pd(C) in 50 mL of MeOH. This afforded 0.76 g (100%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenyalaninate as a thick transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 10 mg of the product with excess 1M ethereal HCl and concentrating to dryness.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 9.05 (d, 1H, J=7.5, NH), 8.24 (br s, 3H, NH₃ ⁺), 7.20 (m, 3H, aromatic CH), 7.07 (t, 1H, J=7.1, aromatic CH), 4.28 (m, 1H, methine), 3.83 (m, 1H, methine), 3.08 (m, 1H, ArCH₂), 2.91 (m, 1H, ArCH₂), 1.78 (m, 1H, cyclohexyl methine), 2.34 (m, 2H, glu 4-CH₂), 2.00 (m, 2H, glu 3-CH₂), 1.89-1.60 (m, 5H, cyclohexyl CH₂), 1.57-1.20 (m, 5H, cyclohexyl CH₂), 1.40 (s, 9H, t-Bu), 1.33 (s, 9H, t-Bu).

EXAMPLE 87 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenylalaninate

According to example 49, 0.247 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.35 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenyalaninate using 0.30 mL of DECP (Aldrich) and 0.32 mL of Et₃ N in 25 mL of DMF. The crude product was subjected to flash chromatography twice (150 g SiO₂, 7:7:1. CH₂ Cl₂ :acetone:MeOH; 150 g SiO₂, 95:5 CH₂ Cl₂ :MeOH) to afford 0.297 g (57%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenylalaninate as a yellow powder, m.p. 135-140° C.

EXAMPLE 88 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclohexyl-L-phenylalanine

According to example 50, 0.271 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-2-cyclohexyl-L-phenylalaninate was treated with gaseous HCl in 20 mL of CH₃ NO₂ for 1.5 h. Work-up and isolation in the usual manner afforded 0.191 g (79%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclohexyl-L-phenylalanine as a yellow-orange powder, m.p. 175° C. (dec).

HPLC: one peak on C 18, k'=4.05, 65:35:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.75-11.90 (br s, COOH), 8.58 (s, 1H pteridinyl 7-CH), 8.20 (d, 1H, J=7.9, amide NH), 7.98 (d, 1H, J=8.1, amide NH), 7.79 (br s, 1H, NH₂), 7.72 (d, 2H, J=8.9, aromatic CH), 7.59 (br s, 1H, NH₂), 7.19 (m, 1H, aromatic CH), 7.08 (m, 2H, aromatic CH), 6.92 (m, 1H, aromatic CH), 6.83 (d, 2H, J=8.9, aromatic CH), 6.75 (br s, 2H, NH₂), 4.80 (s, 2H, pteridinyl-CH₂), 4.43 (m, 1H, methine), 4.31 (m, 1H, methine), 3.21 (s, 3H, N-CH₃), 3.12 (m, 1H, ArCH₂), 3.,92-3.70 (m, 2H, ArCH₂, cyclohexyl methine), 2.23 (t, 2H, J=7.7, glu 4-CH₂), 2.05-1.60 (m, 7H, glu 3-CH₂, cyclohexyl CH₂), 1.50-1.16 (m, 5H, cyclohexyl CH₂).

Elemental Analysis: Calcd. for C₃₅ H₄₁ N₉ O₆ •1.5 H₂ O (MW 710.79): C, 59.14; H, 6.24; N, 17.74. Found: C, 59.11; H, 6.20; N, 17.73.

Mass Spectrum: (FAB) 684 (M+H)⁺, 613, 510, 385, 309.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 259.7 (22300), 308.8 (22700), 374.0 (6850). λ_(min) (ε) 241.7 (12800), 275.2 (14700), 346.3 (5170).

EXAMPLE 89 3-tert-Butylbenzyl bromide

A dry 100 mL 3-necked flask equipped with a nitrogen inlet, a condenser, a thermometer, and a magnetic stirrer was charged with 2.0 g of 3-tert-butyltoluene (Wiley Organics, Coshocton, Ohio 43812), 2.40 g of N-bromosuccinimide (Aldrich), and 30 mL of CCl₄. The mixture was treated with 0.16 g of benzoyl peroxide (Aldrich) and heated at 6-° C. under N₂. Careful temperature control is important to minimize by-product formation. After 1 h at 60° C. the reaction mixture was cooled to 0° C., filtered to remove succinimide, and the filtrate was concentrated in vacuo to afford a light yellow liquid. Analysis of this material by tlc (SiO₂, hexane) indicated a major new component at R_(f) =0.50, a minor component at R_(f) =0.55, and a trace of 3-tert-butyltoluene at R_(f) =0.75. The crude product was purified by flash chromatography on 100 g of silica gel (hexane) to afford 1.06 g (35%) of 3-tert-butylbenzyl bromide as a clear liquid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.47 (s, 1H, aromatic CH), 7.39-7.21 (m, 3H, aromatic CH), 4.69 (s, 2H, ArCH₂ Br), 1.27 (s, 9H, t-Bu).

EXAMPLE 90 (2R, 3S, 5S)-tert-Butyl 3-(3-tert-butylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate

According to example 81, 1.63 g of tert-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich) was alkylated with 1.0 g of 3-tert-butylbenzyl bromide in 70 mL of anhydrous THF using 4.62 mL of 1M sodium bis(trimethylsilyl)amide in THF (Aldrich). The crude product was purified by flash chromatography on 150 g of silica gel (9:1 hexane:EtOAc) to afford 1.84 g (80%) of (2R, 3S, 5S)-tert-butyl 3-(3-tert-butylbenzyl)-2-oxo-2,3-diphenyl-4-morpholinecarboxylate as a white foam, m.p. 65-90° C.

EXAMPLE 91 (2R, 3S, 5S)-6-Oxo-2,3-diphenyl-5-(3-tert-butylbenzyl)morpholine

According to example 82, 1.79 g of (2R, 3S, 5S)-tert-butyl 3-(3-tert-butylbenzyl)-2-oxo-5,6-diphenyl-4-morpholinecarboxylate was treated with 3 mL of TFA in 30 mL of CH₂ Cl₂ for 3.4 h. Neutralization with 7.7 mL of Et₃ N and work-up in the usual manner gave a thick oil. Purification of the crude material by flash chromatography on 125 g of silica gel (75:25 hexane:EtOAc) afforded 0.90 g (63%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butylbenzyl)morpholine as a white crystalline solid, m.p. 136-137° C.

EXAMPLE 92 3-tert-Butyl-L-phenylalanine

According to example 53, 0.89 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butylbenzyl)morpholine was hydrogenolyzed in 40 mL of 1:1 THF:EtOH using 0.28 g of PdCl₂ to afford 0.57 g (99%) of 3-tert-butyl-L-phenylalanine•HCl as a white powder, m.p. ≧250° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.42 (br s, 3H, NH₃ ⁺), 7.26 (m, 3H, aromatic CH), 7.08 (dd; 1H; J=7.0, 1.3; aromatic CH), 4.12 (m, 1H, methine), 3.12 (d, 2H, J=6.1, ArCH₂), 1.27 (s, 9H, t-Bu).

EXAMPLE 93 tert-Butyl 3-tert-butyl-L-phenylalaninate

According to example 54, 0.50 g of 3-tert-butyl-L-phenylalanine•HCl was treated with 30 mL of isobutylene in 25 mL of 1,4-dioxane in the presence of 0.4 mL of conc. H₂ SO₄. Work-up in the usual manner followed by acidification of the free amine with ethereal HCl afforded 0.53 g (87%) of tert-butyl 3-tert-butyl-L-phenylalaninate•HCl as a light yellow foam.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.48 (br s, 3H, NH₃ ⁺), 7.36-7.20 (m, 3H, aromatic CH), 7.08 (d, 1H, J=7.1, aromatic CH), 4.16 (m, 1H, methine), 3.19 (dd; 1H; J=13.9, 5.7; ArCH₂), 2.95 (dd; 1H; J=13.9, 8.6; ArCH₂), 1.28 (s, 9H, t-Bu), 1.27 (s, 9H, t-Bu).

EXAMPLE 94 tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate

According to example 9, 0.52 g of tert-butyl-3-tert-butyl-L-phenylalaninate•HCl was coupled with 0.56 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.33 g of EDC (Aldrich) and 0.18 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 110 g of silica gel (75:25 hexane:EtOAc) to afford 0.77 g (78%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate as a thick transparent oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.28 (d, 1H, J=7.3, NH), 7.42-7.13 (m, 9H, aromatic CH, NH), 7.04 (d, 1H, J=6.6, aromatic CH), 5.02 (s, 2H, PhCH₂ O), 4.37 (m, 1H, methine), 4.07 (m, 1H, methine), 2.96 (d, 2H, J=7.6, ArCH₂), 2.21 (t, 2N, J=7.7. glu 4-CH₂), 1.97-1.60 (m, 2H, glu 3-CH₂), 1.39 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu), 1.27 (s, 9H, t-Bu).

EXAMPLE 95 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate

According to example 48, 0.76 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate was hydrogenolyzed using 0.13 g of 10% Pd(C) in 60 mL of MeOH. This afforded 0.58 g (99%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 10 mg of the product with excess 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.97 (d, 1H, J=7.0, NH), 8.27 (br s, 3H, NH₃ ⁺), 7.31-7.15 (m, 3H, aromatic CH), 7.06 (d, 1H, J=6.8, aromatic CH), 4.38 (m, 1H, methine), 3.82 (m, 1H, methine), 2.96 (d, 2H, J=7.2, ArCH₂), 2.33 (m, 2H, glu 4-CH₂), 1.98 (m, 2H, glu 3-CH₂), 1.38 (s, 9H, t-Bu), 1.29 (s, 9H, t-Bu), 1.26 (s, 9H, t-Bu).

EXAMPLE 96 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate

According to example 49, 0.223 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.30 g of tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate using 0.27 mL of DECP (Aldrich) and 0.29 mL of Et₃ N in 25 mL of DMF. The crude product was subjected to flash chromatography twice (180 g of SiO₂, 7:7:1 CH₂ Cl₂ :acetone:MeOH; 185 g SiO₂, 95:5 CH₂ Cl₂ :MeOH) to afford 0.323 g (71%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate as a yellow powder, m.p, 128-135° C.

EXAMPLE 97 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-phenylalanine (Procedure A)

For Procedure B to prepare this compound, see Example 130. According to example 50, 0.303 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate was treated with gaseous HCl in 40 mL of CH₃ NO₂ for 1.5 h. Work-up and isolation in the usual manner afforded 0.215 g (79%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-phenylalanine as a yellow powder, m.p. 183° C.

HPLC: one peak on C18, k'=2.54, 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.90-11.80 (br s, COOH), 8.59 (s, 1H, pteridinyl 7-CH), 8.08 (d, 1H, J=7.6, amide NH), 7.98 (d, 1H, J=8.0, amide NH), 7.89 (br s, 1H, NH₂), 7.70 (d, 2H, J=8.9, aromatic CH), 7.69 (br s, 1H, NH₂), 7.21 (s, 1H, aromatic CH), 7.16 (d, 1H, J=7.7, aromatic CH), 7.08 (t, 1H, J=7.6, aromatic CH), 6.99 (d, 1H, J=7.4, aromatic CH), 6.81 (m, 4H, aromatic CH, NH₂), 4.79 (s, 2H, pteridinyl CH₂), 4.43 (m, 2H, methines), 3.21 (s, 3H, N-CH₃), 3.03 (m, 1H, ArCH₂), 2.91 (m, 1H, ArCH₂), 2.24 (t, 2H, J=7.8 glu 4-CH₂), 2.04-1.74 (m, 2H, glu 3-CH₂), 1.20 (s, 9H, t-Bu).

Elemental Analysis: Calcd. for C₃₃ H₃₉ N₉ O₆ •1.8 H₂ O (MW 690.16): C, 57.43; H, 6.22; N, 18.27. Found: C, 57.34; H, 6.07; N, 18.26.

Mass Spectrum: (FAB) 658 (M+H)⁺, 459, 307.

UV Spectrum: (pH7 Buffer) λ_(max) (ε) 259.4 (22800), 308.1 (23000), 372.5 (7040). λ_(min) (ε) 241.0 (13400), 274.6 (15200), 345.2 (5380).

EXAMPLE 98 N-((tert-Butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)-L-phenylalanine

A dry 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.10 g of 4'-(bis(2-chloroethyl)amino)-L-phenylalanine•H₂ O (Sigma), 14 mL of 1:1 DMF:CH₃ CN, and 0.19 mL of Et₃ N. The mixture was treated with 91 mg of 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON, Fluka) and stirred at RT under N₂. After 18 h, the solid starting material had dissolved giving a light yellow solution which was concentrated in vacuo to dryness. Analysis of the residue by tlc (SiO₂, 9:1 CHCl₃ :MeOH) indicated a major new component at R_(f) =0.65, BOC-ON derived components at R_(f) =1.0 and 0.71 and two trace components at R_(f) =0.60 and 0.35. The crude product was purified by flash chromatography on 60 g of silica gel (9:1 CHCl₃ :MeOH) to afford 0.10 g (80%) of N-((tert-butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)-L-phenylalanine as light yellow glass.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.04 (d, 2H, J=8.6, aromatic CH), 6.62 (d, 2H, J=8.5, aromatic CH), 6.46 (br s, 1H, NH), 3.87 (m, 1H, methine), 3.67 (s, 8H, N (CH₂ CH₂ Cl)₂), 2.90 (m, 1H, ArCH₂), 1.33 (s, 9H, t-Bu).

EXAMPLE 99 tert-Butyl N-((tert-butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)-L-phenylalanyl-(3-cyclopentyl)-L-phenylalaninate

A 50 mL round bottomed flask equipped with a magnetic stirrer was charged with 96 mg of N-((tert-butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)-L-phenylalanine 77 mg of tert-butyl 3-cyclopentyl-L-phenylalaninate•HCl, and 10 mL of CH₂ Cl₂. The solution was cooled to 0° C. and treated with 48 mg of EDC (Aldrich) followed by 0.026 mL of N-methylmorpholine. The reaction mixture was stirred at 0° C. for 20 min, allowed to war to RT, and stirred for an additional 2 h. Analysis of the solution by tlc (SiO₂, 7:3 hexane:EtOAc) indicated a major new component at R_(f) =0.54 and four minor components at R_(f) =0.4, 0.37, 0.16 and 0.0. The solution was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on 50 g of silica gel to afford 73 mg (46%) of tert-butyl-N-((tert-butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)-L-phenylalanyl-(3-cyclopentyl)-L-phenylalaninate as a thick semi-solid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.18 (d, 1H, J=7.8, NH), 7.19 (m, 1H, aromatic CH), 7.13-7.00 )m, 5H, aromatic CH, NH), 6.77 (d, 1H, J=8.8, aromatic CH), 6.63 (d, 2H, J=8.9, aromatic CH), 4.35 (m, 1H, methine), 4.05 (m, 1H, methine), 3.68 (s, 8H, N (CH₂ CH₂ Cl)₂), 2.93 (m, 3H, ArCH₂, cyclopentyl methine), 2.79 (m, 1H, ArCH₂), 2.58 (m, 1H, ArCH₂), 1.80-1.43 (m, 8H, cyclopentyl CH₂), 1.29 (s, 18H, t-Bu).

EXAMPLE 100 4-(Bis(2-chloroethyl)amino)-L-phenylalanyl-3-cyclopentyl-L-phenylalanine

A 25 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 65 mg of tert-butyl-N-((tert-butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)-L-phenylalanyl-(3-cyclopentyl)-L-phenylalaninate and 8 mL of CH₃ NO₂. The solution was acidified by bubbling HCl gas through for 10 min. After stirring at RT under N₂ for 30 min, the solution was concentrated in vacuo to dryness. Analysis of the resulting solid by HPLC (C18, 50:50:0.1 H₂ O:MeCN:TFA) indicated a major component at k'=1.14 (96%) and a minor component at k'=1.60 (4%). The crude product was purified by semi-preparative HPLC (C18, 55:45:0.1→50:50:0.1 H₂ O:MeCN:TFA over 20 min, flow rate=15 mL/min). Fractions containing pure product (k'=1.14 by analytical HPLC) were combined and concentrated in vacuo to 20 mL. The resulting suspension was frozen and lyophilized to afford 35 mg (55%) of 4-(bis(2-chlorethyl)amino)-L-phenylalanyl-3-cyclopentyl-L-phenylalanine as a white powder, m.p. 80-90° C.

HPLC: one peak on C 18, k'=1.05, 50:50:0.1 MeCN•H₂ O:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.85 (d, 1H, J=7.8, NH), 8.00 br s, 3H, NH₃ ⁺), 7.25-7.00 (m, 6H, aromatic CH), 6.70 (d, 2H, J=8.5, aromatic CH), 4.50 (m, 1H, methine), 3.89 (m, 1H, methine), 3.69 (5, 8H, N (CH₂ CH₂ Cl)₂), 3.12-2.83 (m, 4H, ArCH₂, cyclopentyl methine), 2.77 (m, 1H, ArCH₂), 1.97 (m, 2H, cyclopentyl CH₂), 1.84-1.43 (m, 6H, cyclopentyl CH₂).

¹⁹ F-NMR: (282 MHz, DMSO-d₆): d 1.26 (relative to TFA external standard).

Elemental Analysis: Calcd. for C₂₇ H₃₅ N₃ O₃ Cl₂ •TFA•1.6 H₂ O (MW 663.35): C, 52.51; H, 5.96; N, 6.33. Found: C, 52.16; H, 5.69; N, 6.69.

Mass Spectrum: (FAB) 520 (M+H)⁺, 273.

UV Spectrum: (2.5×10⁻³ M NaOH) λ_(max) (ε) 261.5 (18700), 303.9 (2090), λ_(min) (ε) 232.3 (5270), 288.2 (1760).

EXAMPLE 101 3-Iodo-O-(tert-butyldimethylsilyl)benzyl alcohol

A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 5.0 g of 3-iodobenzyl alcohol (Aldrich), 3.86 g of tert-butyldimethylsilyl chloride (Aldrich), 3.20 g of imidazole (Aldrich), and 30 mL of anhydrous DMF. The resulting solution was stirred under nitrogen for 4 h and concentrated in vacuo to give a clear viscous oil. This material was dissolved in 150 mL of EtOAc and the solution was washed with 5% aqueous NaHCO₃ (3×70 mL), dried over anhydrous MgSO₄ and concentrated in vacuo to give a clear liquid. The crude product was purified by flash chromatography on 200 g of silica gel (hexane) to afford 7.01 g (94%) of 3-iodo-O-(tert-butyldimethylsilyl)benzyl alcohol as a clear liquid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.67 (s, 1H, aromatic CH), 7.56 (d, 1H, J=7.3, aromatic CH), 7.27 (d, 1H, J=9.2, aromatic CH), 7.06 (t, 1H, J=7.6, aromatic CH), 4.68 (s, 2H, ArCH₂ O), 0.94 (s, 9H, t-Bu), 0.10 (s, 6H, SiMe₂).

EXAMPLE 102 3-(1-Hydroxycyclobutyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol

T a dry 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer were added 7.0 g of 3-iodo-O-(tert-butyldimethylsilyl)benzyl alcohol and 100 mL of anhydrous THF. The solution was cooled to -78° C. in a dry ice-isopropanol bath and treated with 9.7 mL of 2.5M n-butyllithium/hexane (Aldrich) by dropwise addition via syringe through a rubber septum. After stirring at -78° C. for 20 min, the solution was treated with 1.80 mL of cyclobutanone (Aldrich) by dropwise addition. The reaction mixture was stirred at -78° C. for 20 min and then allowed to warm to RT. After 30 min at RT the solution was mixed with 100 mL of water an subjected to rotary evaporation to remove THF. The resulting emulsion was extracted with EtOAc (4×60 mL). The combined EtOAc extracts were washed with saturated brine, dried over anhydrous MgSO₄, and concentrated in vacuo to give a light yellow oil. This material was purified by flash chromatography on 160 g of silica gel (9:1 hexane:EtOAc) to afford 3.98 g (68%) of 3-(1-hydroxycyclobutyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol as a clear liquid.

¹ H-NMR: (300 MHz, DMSO-d₆) d 7.44 (s, 1H, aromatic CH), 7.38-7.23 (m, 2H, aromatic CH), 7.15 (d, 1H, J=7.4, aromatic CH), 5.45 (s, 1H, OH), 4.72 (s, 2H, ArCH₂ O), 2.41-2.18 (m, 4H, cyclobutyl CH₂), 1.88 (m, 1H, cyclobutyl CH₂), 1.61 (m, 1H, cyclobutyl CH₂), 0.90 (s, 9H, t-Bu), 0.08 (s, 6H, SiMe₂).

EXAMPLE 103 3-Cyclobutylbenzyl alcohol

A dry 500 mL 3-necked flask equipped with a nitrogen inlet and magnetic stirrer was charged with 3.98 g of 3-(1-hydroxycyclobutyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol and 85 mL of CH₂ Cl₂. The solution was cooled to 0° C. under nitrogen and treated with 15.7 mL of TFA followed by 4.78 mL of triethylsilane. After stirring at 0° C. for 1 h, the solution was allowed to warm to RT. Analysis by tlc (SiO₂, 8:2 hexane:EtOAc) after 3.5 h at RT indicated unreacted starting material at R_(f) =0.15 and a new component at R_(f) =0.65. The solution was treated with an additional 3 mL of triethylsilane and stirred at RT for 18 h. The solution was cooled to 0° C. and neutralized by slow addition of 30 mL of Et₃ N. The mixture was concentrated invacuo to give a light yellow liquid which was dissolved in 100 mL of EtOAc. The resulting solution was washed with saturated brine (3×80 mL), dried over anhydrous MgSO₄, and concentrated to give a clear liquid. This material was dissolved in 80 mL of anhydrous THF and treated with 60 mL of 1M (n-Bu)₄ NF (Aldrich) in a 500 mL of 3-necked flask. After stirring at RT for 18 h, the solution was concentrated in vacuo and the residue dissolved in 100 mL of EtOAc. The solution was washed with saturated brine (4×80 mL), dried over anhydrous MgSO₄, and concentrated to give a clear oil. The crude product was purified by flash chromatography on 120 g of silica gel (8:2 hexane:EtOAc) to afford 1.29 g (59%) of 3-cyclobutylbenzyl alcohol as a clear liquid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.29-7.05 (m, 4H, aromatic CH), 5.24 (t, J=5.7, OH), 4.48 (d, J=5.8, ArCH₂ O), 3.51 (m, 1H, cyclobutyl methine), 2.30 (m, 2H, cyclobutyl CH₂), 2.17-1.88 (m, 3H, cyclobutyl CH₂), 1.82 (m, 1H, cyclobutyl CH₂).

EXAMPLE 104 3-Cyclobutylbenzyl bromide

According to example 17, 1.29 g of 3-cyclobutylbenzyl alcohol was treated with 0.26 mL of PBr₃ in 15 mL of anhydrous CH₂ Cl₂ to afford 1.69 g of crude product (92% pure by 1H-NMR). This material was purified by flash chromatography on 65 g of silica gel (hexane) to afford 1.59 g (89%) of 3-cyclobutyl benzyl bromide as a clear liquid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.35-7.13 (m, 4H, aromatic CH), 4.70 (s, 2H, ArCH₂ Br), 3.53 (m, 1H, cyclobutyl methine), 2.39-1.75 (m, 6H, cyclobutyl CH₂).

EXAMPLE 105 tert-Butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-cyclobutylbenzyl)-4-morpholinecarboxylate

According to example 81, 2.50 g of tert-butyl- (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich) was alkylated with 1.59 g of 3-cyclobutylbenzyl bromide in 80 mL of anhydrous THF using 7.41 mL of 1M sodium bis(trimethylsilyl)amide in THF (Aldrich). The crude product was subjected to flash chromatography on 120 g of silica gel (8:2 hexane:EtOAc) and then recrystallization from EtOH-water to afford 1.90 g (54%) of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-cyclobutylbenzyl)-4-morpholinecarboxylate as a white powder, m.p. 145-147° C.

EXAMPLE 106 (2R, 3S, 5S)-6-Oxo-2,3-diphenyl-5-(3-cyclobutylbenzyl)morpholine

According to example 82, 1.87 g of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-cyclobutylbenzyl)-4-morpholinecarboxylate was treated with 3.1 mL of TFA in 30 mL of CH₂ Cl₂ for 4 h. Neutralization with 8 mL of Et₃ N and work-up in the usual manner gave a light yellow residue. Purification of the crude material by flash chromatography on 85 g of silica gel (8:2 hexane:EtOAc) afforded 1.35 g (91%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-cyclobutylbenzyl)morpholine as a thick, transparent semi-solid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.24-7.02 (m, 10H, aromatic CH), 6.92 (m, 4H, aromatic CH), 5.71 (s, 1H, morpholine 2-H), 4.64 (m, 1H, morpholine 3-H), 4.24 (m, 1H, morpholine 5-H), 3.42 (m, 1H, cyclobutyl methine), 3.26 (m, 1H, ArCH₂), 3.10 (m, 1H, ArCH₂), 2.76 (t, 1H, J=5.3, NH), 2.15 (m, 2H, cyclobutyl CH₂), 2.02-2.81 (m, 3H, cyclobutyl CH₂), 1.71 (m, 1H, cyclobutyl CH₂).

EXAMPLE 107 3-Cyclobutyl-L-phenylalanine

According to example 53, 1.35 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-cyclobutylbenzyl)morpholine was hydrogenolyzed in 60 mL of 1:1 THF:EtOH using 0.42 g of PdCl₂ to afford 0.79 g (91%) of 3-cyclobutyl-L-phenylalanine•HCl as a white powder, m.p. ≧200° C.

¹ H-NMR: (300 MHz-DMSO-d₆) δ 8.40(br s, 3H, NH₃ ⁺), 7.26 (m, 1H, aromatic CH), 7.13 (m, 2H, aromatic CH), 7.08 (d, 1H, J=7.4, aromatic CH), 4.13 (m, 1H, methine), 3.50 (m, 1H, cyclobutyl methine), 3.11 (d, 2H, J=7.4, ArCH₂), 2.29 (m, 2H, cyclobutyl CH₂), 2.18-1.88 (m, 3H, cyclobutyl CH₂), 1.83 (m, 1H, cyclobutyl CH₂).

EXAMPLE 108 tert-Butyl 3-cyclobutyl-L-phenylalaninate

According to example 54, 0.65 g of 3-cyclobutyl-L-phenylalanine•HCl was treated with 25 mL of isobutylene in 25 mL of 1,4-dioxane in the presence of 0.5 mL of conc. H₂ SO₄. Work-up in the usual manner followed by acidification of the free amine with ethereal HCl afforded 0.64 g (81%) of tert-butyl 3-cyclobutyl-L-phenylalaninate•HCl as a white powder, m.p. 173-174° C.

EXAMPLE 109 tert-Butyl N-(benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate

According to example 9, 0.64 g of tert-butyl 3-cyclobutyl-L-phenylalaninate•HCl was coupled with 0.69 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.41 g of EDC (Aldrich) and 0.23 mL of N-methylmorpholine. The crude product was purified by flash cromatography on 85 g of silica gel (7:3 hexane:EtOAc) to afford 0.98 g (80%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate as a thick transparent oil.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.21 (d, 1H, J=7.3, NH), 7.33 (m, 6H, aromatic CH, NH), 7.18 (m, 1H, aromatic CH), 7.02 (m, 3H, aromatic CH), 5.00 (m, 2H, PhCH₂ O), 4.32 (m, 1H, methine, 4.05 (m, 1H, methine), 3.47 (m, 1H, cyclobutyl methine), 2.92 (d, 2H, J=7.6, ArCH₂), 2.31-2.12 (m, 4H, glu 4-CH₂, cyclobutyl CH₂), 2.11-1.62 (m, 6H, glu 3-CH₂, cyclobutyl CH₂), 1.37 (s, 9H, t-Bu), 1.29 (s, 9H, t-Bu).

EXAMPLE 110 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate

According to example 48, 0.98 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate was hydrogenolyzed using 0.17 g of 10% Pd(C) in 55 mL of MeOH. This afforded 0.76 g (100%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 10 mg of the product with excess 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.92 (d, 1H, J=7.0, NH), 8.25 (br s, 3H, NH₃ ⁺), 7.20 (m, 1H, aromatic CH), 7.08 (m, 3H, aromatic CH), 4.38 (m, 1H, methine), 3.83 (m, 1H, methine), 3.48 (m, 1H, cyclobutyl methine), 2.94 (d, 2H, J=7.8, ArCH₂), 2.16 (m, 4H, glu 4-CH₂, cyclobutyl CH₂), 2.15-1.86 (m, 5H, glu 3-CH₂, cyclobutyl CH₂), 1.79 (m, 1H, cyclobutyl CH₂), 1.38 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu).

EXAMPLE 111 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate

According to example 49, 0.285 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.38 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate using 0.34 mL of DECP (Aldrich) and 0.37 mL of Et₃ N in 25 mL of DMF. The crude product was subjected to flash chromatography on 100 g of silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.45 g (78%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate as a yellow powder, m.p. 120-125° C.

EXAMPLE 112 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclobutyl-L-phenylalanine

According to example 50, 0.40 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclobutyl-L-phenylalaninate was treated with gaseous HCl in 60 mL of CH₃ NO₂ for 1 h. Work-up and isolation in the usual manner afforded 0.281 g (79%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclobutyl-L-phenylalanine as a yellow powder, m.p. 175° C. (dec).

HPLC: one peak on C18, k'=2.50, 65:35:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.85-11.75 (br m, COOH), 8.58 (s, 1H, pteridinyl 7-CH), 8.04 (d, 1H, J=8.1, amide NH), 8.00 (d, 1H, J=8.7, amide NH), 7.75 (br s, 1H, NH₂), 7.71 (d, 2H, J=8.9, aromatic CH), 7.55 (br s, 1H, NH₂), 7.10-6.92 (m, 4H, aromatic CH), 6.82 (d, 2H, J=8.9, aromatic CH), 6.70 (br s, 2H, NH₂), 4.79 (s, 2H, pteridinyl-CH₂), 4.39 (m, 2H, methines), 3.40 (m, 1H, cyclobutyl methine), 3.21 (s, 3H, N-CH₃), 3.00 (m, 1H, ArCH₂), 2.88 (m, 1H, ArCH₂), 2.29-2.08 (m, 4H, glu 4-CH₂, cyclobutyl CH₂), 2.07-1.65 (m, 6H, glu 3-CH₂, cyclobutyl CH₂).

Elemental Analysis: Calcd. for C₃₃ H₃₇ N₉ O₆ •1.6 H₂ O (MW 684.54): C, 57.90; H, 5.92; N, 18.42. Found: C, 57.88; H, 5.90; N, 18.52.

Mass Spectrum: (Ion Spray) 656 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 259.5(22100), 308.0 (22300), 373.7 (6740). λ_(min) (ε) 241.2 (12900), 274.5 (14600), 345.6 (5130).

EXAMPLE 113 6-Bromomethyl-3,4-dihydro-2-methyl-4-oxoquinazoline

According to example 4, 1.00 g of 3,4-dihydro-2,6-dimethyl-4-oxoquinazoline (Sen, A. B.; Gupta, J. K. J. Indian Chem. Soc., 1962, 31, 369) was subjected to bromination using 1.05 g of N-bromosuccinimide (Aldrich) and 0.17 g of benzoyl peroxide in 50 mL of 1,2-dichloro-ethane for 30 min to afford 0.95 g (64%) of 6-bromomethyl-3,4-dihydro-2-methyl-4-oxoquinazoline as a tan solid.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 8.17 (s, 1H, aromatic CH), 7.85 (d, 1H, J=8.5, aromatic CH), 7.58 (d, 1H, J=8.5, aromatic CH), 4.88 (s, 2H, ArCH₂ Br), 2.39 (s, 3H, CH₃).

EXAMPLE 114 Ethyl 5-N-methylamino-2-thiophenecarboxylate

A mixture of 2.0 g of ethyl 5-amino-2-thiophenecarboxylate (Mackay, D. Can. J. Chem., 1966, 44, 2881-2891; Paul,H.; Migulla, H. Arch. Pharm., 1978, 311, 679-691.), 2.05 g of methyl iodide (Aldrich), and 2.58 g of 2,6-lutidine (Aldrich) in 80 mL of DMF was stirred at 70° C. under nitrogen for 24 h. Analysis of the reaction mixture by tlc (SiO₂, 3:2 hexane:EtOAc) indicated starting material at R_(f) =0.51 and a new component at R_(f) =0.60. The mixture was treated with an additional 1.71 g of methyl iodide and was stirred at 70° C. for 8 h, RT for 72 h and 70° C. for an additional 8 h. The reaction mixture was mixed with 75 mL of water and extracted with EtOAc (3×150 mL). the combined EtOAc extracts were washed with saturated aqueous brine, dried over anhydrous Na₂ SO₄ and concentrated in vacuo to dryness. The crude residue was subjected to flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 0.67 g (38%) of ethyl 5-N-methylamino-2-thiophenecarboxylate as brown solid, m.p. 41-43° C.

EXAMPLE 115 Ethyl 5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thiophene-carboxylate

A 100 mL round bottomed flask equipped with a condenser and magnetic stirrer was charged with 0.66 g of 6-bromomethyl-3,4-dihydro-2-methyl-4-oxoquinazoline, 0.44 g of ethyl 5-N-methylamino-2-thiophenecarboxylate, 0.25 g of 2,6-lutidine, and 30 mL of anhydrous DMF. The mixture was stirred at 80° C. for 18 h. Analysis of the reaction mixture by tlc (SiO₂, 95:5CH₂ Cl₂ :MeOH) indicated starting materials and a new component at R_(f) 32 0.41. The mixture was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on 110 g of silica gel (95:5 CH₂ Cl₂ :MeOH). Fractions containing the R_(f) =0.41 component were combined and concentrated to afford 0.42 g (49%) of ethyl 5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thiophenecarboxylate as a light tan powder, m.p. ≧200° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.22 (br s, 1H, NH), 7.92 (s, 1H, aromatic CH), 7.65 (d, 1H, J=8.4, aromatic CH), 7.56 (d, 1H, J=8.5, aromatic CH), 7.48 (d, 1H, J=4.4, thienyl CH), 6.07 (d, 1H, J=4.3, thienyl CH), 4.70 (s, 2H, quinazolinyl-CH₂), 4.16 (q, 2H, J=7.2, ethyl CH₂), 3.09 (s, 3H, N-CH₃), 2.33 (s, 3H, quinazolinyl 2-CH₃), 1.22 (t, 3H, J=7.2, ethyl CH₃).

EXAMPLE 116 5-(((3,4-Dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thiophene-carboxylic acid

A 25 mL 3-necked flask equipped with a condenser, a thermometer, and a magnetic stirrer was charged with 0.20 g of ethyl 5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thiophene-carboxylate, 6 mL of EtOH, and 6 mL of 1N aqueous NaOH. The resulting solution was stirred under nitrogen and RT for 25 min and then at 55° C. for 2 h. Analysis by tlc (SiO₂, 95:5 CH₂ Cl₂) indicated no remaining starting material and a new component at R_(f) =0.06. The solution was cooled to RT and subjected to rotary evaporation to remove EtOH. The remaining solution was diluted with 10 mL of water and acidified to pH=3.5 by addition of 1N aqueous HCl. A tan precipitate formed which was collected by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.116 g (63%) of 5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thiophene-carboxylic acid as a white powder, m.p. 195° C. (dec).

¹ H-NMR: (200 MHz, DMSO-d₆) δ 12.40-11.90 (br m, COOH, NH), 7.95 (s, 1H, aromatic CH), 7.69 (m, 1H, aromatic CH), 7.58 (d, 1H, J=8.2, aromatic CH), 7.42 (d, 1H, J=4.2, thienyl CH), 6.04 (d, 1H, J=4.2, thienyl CH), 4.70 (s, 2H, quinazolinyl-CH₂), 3.09 (s, 3H, N-CH₃), 2.35 (s, 3H, quinazolinyl 2-CH₃).

EXAMPLE 117 tert-Butyl N-((5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

A dry 50 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 0.115 g of 5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thiophene-carboxylic acid, 0.165 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate 0.06 mL of Et₃ N, and 7 mL of anhydrous DMF. The yellow solution was treated with 0.06 mL of DECP and was stirred at RT under nitrogen. After 2 h, tlc (SiO₂, 95:5 CH₂ Cl₂ :MeOH) indicated starting material at R_(f) =0.02, a major new component at R_(f) =0.37, and minor components at R_(f) =0.31 and 0.40. The reaction mixture was treated with an additional 0.03 mL of DECP and 0.03 mL of Et₃ N and was stirred for another 1 h at RT. TLC indicated little additional conversion. The solution was concentrated in vacuo to dryness and the residue dissolved in 60 mL of CHCl₃. The solution was washed with 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous MgSO₄, and concentrated to give a light yellow oil. The crude product was subjected to flash chromatography twice (SiO₂, 95:5 EtOAc:MeOH; SiO₂, 97:3 CH₂ Cl₂ :MeOH) to afford 0.119 g (46%) of tert-butyl N-((5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a tan foam.

¹ H-NMR: (400 MHz, DMSO-d₆) δ 12.19 (s, 1H, quinazoline NH), 8.18 (d, 1H, J=7.1, amide NH), 7.92 (m, 2H, amide NH, aromatic CH), 7.63 (dd; 1H; J=7.9, 1.8; aromatic CH), 7.57 (d, 1H, J=4.1, thienyl CH), 7.54 (d, 1H, J=8.0, aromatic CH), 7.12-6.94 (m, 4H, aromatic CH), 5.97 (d, 1H, J=4.1, thienyl CH), 4.63 (s, 2H, quinazolinyl-CH₂), 4.40-4.27 (m, 2H, methines), 3.02 (s, 3H, N-CH₃), 2.87 (m, 3H, ArCH₂, cyclopentyl methine), 2.31 (s, 3H, quinazolinyl 2-CH₃), 2.20 (t, 2H, J=7.7, glu 4-CH₂), 1.91 (m, 3H, glu 3-CH₂, cyclopentyl CH₂), 1.83-1.40 (m, 7H, cyclopentyl CH₂), 1.35 (s, 9H, t-Bu), 1.27 (s, 9H, t-Bu).

EXAMPLE 118 N-((5-(((3,4-Dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine

According to example 50, 0.117 g of tert-butyl N-((5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate was treated with gaseous HCl in 25 mL of CH₃ NO₂ for 45 min. Removal of the CH₃ NO₂ in vacuo gave a tan residue which was purified by semi-preparative reverse phase HPLC (C18, 70:30:01→60:40:01 H₂ O:MeCN:TFA over 25 min). Fractions containing the major component (k'=1.61 on analytical HPLC, C18, 60:40:0.1 H₂ O:MeCN:TFA) were combined, concentrated to 25 mL and lyophilized to afford 0.052 g (42%) of N-((5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine as a yellow powder, m.p. 125° C. (dec).

HPLC: one peak on C18, k'=1.62, 60:40:0.1 H₂ O:MeCN:TFA, flow rate =1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 8.09 (d, 1H, J=7.5, amide NH), 7.93 (m, 2H, amide NH, aromatic CH), 7.69 (d, 1H, J=8.0, aromatic CH), 7.56 (m, 2H, aromatic CH, thienyl CH), 7.10-6.92 (m, 4H, aromatic CH), 5.97 (d, 1H, J=4.3, thienyl CH), 4.66 (s, 2H, quinazolinyl-CH₂), 4.33 (m, 2H, methines), 3.06 (s, 3H, N-CH₃), 2.99 (m, 1H, ArCH₂), 2.85 (m, 1H, ArCH₂), 2.38 (s, 3H, quinazolinyl 2-CH₃), 2.22 (t, 2H, J=7.5, glu 4-CH₂), 2.02-1.37 (m, 10H, glu 3-CH₂, cyclopentyl CH₂).

¹⁹ F-NMR: (376 MHz, DMSO-d₆) δ 0.57 (relative to TFA external standard).

Elemental Analysis: Calcd. for C₃₅ H₃₉ N₅ O₇ S•1.2 TFA•1.3 H₂ O (MW 834.03): C, 53.86; H, 5.17; N, 8.40; S, 3.84. Found: C, 53.85; H, 5.11; N, 8.36; S, 3.92.

Mass Spectrum: (Ion Spray) 691 (M+NH₄ ⁺).

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 225.2 (36600), 265.6 (10300), 353.1 (22200). λ_(min) (ε) 214.3 (35200), 252.1 (9230), 288.4 (4120).

EXAMPLE 119 tert-Butyl 5-O-tert-butyl-N-(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)-trifluoroacetamido)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

A 25 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.258 g of 4-(N-(3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)trifluoroacetamido)benzoic acid (Styles, V. L., et al, J. Heterocyclic Chem., 1990, 27, 1809), 0.250 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate, 0.077 g of 1-hydroxybenzotriazole hydrate (Aldrich), 5 mL of anhydrous DMF, and 0.07 mL of Et₃ N. The solution was treated with 0.104 g of DCC (Fluka) and stirred at RT under nitrogen for 24 h. The mixture was filtered and the filtrate was concentrated in vacuo to dryness. The residue was dissolved in 70 mL of CH₂ Cl₂, washed with 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous MgSO₄ and concentrated to give a tan oil. This material was purified by flash chromatography on silica gel (95:5 AE 90:10 CH₂ Cl₂ :MeOH) to afford 0.304 g (70%) of tert-butyl 5-O-tert-butyl-N-(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)trifluoroacetamido)-benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a tan glass, m.p. 124-130° C.

EXAMPLE 120 tert-Butyl-5-O-tert-butyl-N-(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

To a 100 mL round bottomed flask equipped with a nitrogen inlet and a magnetic stirrer were added 0.268 g of tert-butyl 5-O-tert-butyl-N-(4-((3-(2,4-diamino-1,6-dihydro-6oxo-5-pyrimidinyl)propyl)trifluoroacetamido)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate, and 7 mL of MeOH. The solution was treated with 0.18 mL of 40% (w/w) dimethylamine/water (Aldrich) followed by 0.45 mL of water and was stirred at RT under nitrogen for 24 h. The solution was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on 60 g of silica gel (92:8 CH₂ Cl₂ :MeOH) to afford 0.185 g (78%) of tert-butyl 5-O-tert-butyl-N-(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a thick transparent semi-solid.

¹ H-NMR: (200 MHz, DMSO-d₆)δ 9.80 (br s, 1H, pyrimidinyl NH), 8.20 (d, 1H, J=7.2, amide NH), 7.90 (d, 1H, J=8.0, amide NH), 7.67 (d, 2H, J=8.6, aromatic CH), 7.20-6.98 (m, 4H, aromatic CH), 6.55 (d, 2H, J=8.6, aromatic CH), 6.25 (m, 1H, NH), 5.94 (br s, 2H, NH₂), 5.77 (br s, 2H, NH₂), 4.54-4.29 (m, 2H, methines), 3.12-2.80 (m, 5H, cyclopentyl methine, ArCH₂, pyrimidinyl-CH₂ CH₂ CH₂ N), 2.27 (m, 4H, glu 4-CH₂, pyrimidinyl-CH₂ CH₂ CH₂ N), 2.10-1.82 (4H, glu 3-CH₂, cyclopentyl CH₂), 1.80-1.45 (m, 8H, cyclopentyl CH₂, pyrimidinyl-CH₂ CH₂ CH₂ N), 1.39 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu).

EXAMPLE 121 N-(4-((3-(2,4-Diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine

A 100 mL round bottomed flask equipped with a magnetic stirrer was charged with 0.180 g of tert-butyl 5-O-tert-butyl-N-(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate and 25 mL of CH₃ NO₂. The mixture was acidified by bubbling HCl gas through for 5 min, however, the solid starting material did not dissolve. Analysis of the reaction mixture by tlc (SiO₂, 92.8 CH₂ Cl₂ :MeOH) indicated minimal conversion of the starting material. The mixture was concentrated in vacuo to dryness. A yellow residue resulted which was suspended in 20 mL of CH₂ Cl₂ and treated with 1 mL of anisole followed by 15 mL of TFA. The solid quickly dissolved giving a yellow solution which was stirred at RT under nitrogen. After 1 h, tlc indicated no remaining starting material and a new spot of R_(f) =0.0. The solution was concentrated to dryness and the residue was purified by semi-preparative reverse phase HPLC (C18, 70:30:0.1 H₂ O: MeCN:TFA). Fractions containing pure material were combined, concentrated to 30 mL and lyophilized to afford 0.098 g (47%) of N-(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine as a fluffy white solid, m.p. 125° C. (dec).

HPLC: one peak on C18, k'=1.64, 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆)δ 12.91-11.10 (br s, COOH, pyrimidinyl NH), 8.07 (d, 1H, J=7.6, amide NH), 7.88 (d, 1H, J=7.9, amide NH), 7.70 (br s, 1H, NH₂), 7.62 (m, 3H, NH₂, aromatic CH), 7.13-6.70 (m, 6H, NH₂, aromatic CH), 6.53 (d, 2H, J=8.6, aromatic CH), 4.40 (m, 2H, methines), 3.01 (m, 3H, cyclopentyl methine, pyrimidinyl-CH₂ CH₂ CH₂ N), 2.86 (m, 2H, ArCH₂), 2.25 (m, 4H, glu 4-CH₂, pyrimidinyl-CH₂ CH₂ CH₂ N), 2.01-1.78 (m, 4H, glu 3-CH₂, cyclopentyl CH₂), 1.76-1.37 (m, 8H, cyclopentyl CH₂, pyrimidinyl-CH₂ CH₂ CH₂ N).

¹⁹ F-NMR: (282 MHz, DMSO-d₆)δ 3.92 (relative to TFA external standard).

Elemental Analysis: Calcd. for C₃₃ H₄₁ N₇ O₇ 1.8 TFA 1.6 H₂ O (MW 881.80): C, 49.85; H, 5.26; N, 11.12. Found: C, 49.81; H, 5.24; N, 11.11.

Mass Spectrum: (Ion Spray) 648 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 280.1 (29700). λ_(min) (ε) 247.4 (7590). sh (e) 302.9 (20400).

EXAMPLE 122 Di-tert-butyl N-(benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-L-asparate

According to example 9, 1.00 g of L-aspartic acid di-t-butyl ester hydrochloride (Sigma) was coupled with 1.20 g of N-Cbz-L-glutamic acid λ-t-butyl ester (Sigma) using 0.72 g of EDC (Aldrich) and 0.39 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 80 g of silica gel (75:25 hexane:EtOAc) to afford 1.10 g (55%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-4-O-tert-butyl-L-aspartate as a white solid, m.p. 119-120° C.

EXAMPLE 123 Di-tert-butyl 5-O-tert-butyl-L-glutam-1-yl-L-asparate

According to example 48, 0.389 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-4-O-tert-butyl-L-aspartate was hydrogenolyzed using 0.07 g of 10% Pd(C) in 40 mL of MeOH. This afforded 0.29 g (98%) of di-tert-butyl 5-O-tert-butyl-L-glutam-1-yl-L-asparate as a thick transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 10 mg of the product with excess 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 8.91 (d, 1H, J=7.8, NH), 8.31 (br s, 3H, NH₃ ⁺), 4.52 (m, 1H, methine), 3.83 (m, 1H, methine), 2.63 (d, 2H, J=6.1, asp CH₂), 2.36 (m, 2H, glu 4-CH₂), 1.94 (m, 2H, glu 3-CH₂), 1.38 (s, 27H, t-Bu).

EXAMPLE 124 Di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-aspartate

According to example 49, 0.30 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.34 g of di-tert-butyl 5-O-tert-butyl-L-glutam-1-yl-L-asparate using 0.36 mL of DECP (Aldrich) and 0.39 mL of Et₃ N in 25 mL of DMF. The crude product was subjected to flash chromatography twice (SiO₂, 7:7:1 CH₂ Cl₂ :acetone:MeOH; SiO₂, 96:4→95:5 CH₂ Cl₂ :MeOH) to afford 0.213 g (37%) of di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-asparate as a yellow powder, m.p. 130-135° C.

EXAMPLE 125 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-aspartic acid (Procedure B)

For Procedure A to prepare this compound, see Example 33.

According to example 50, 0.307 g of di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-asparate was treated with gaseous HCl in 40 mL of CH₃ NO₂ for 1.5 h. The reaction mixture was concentrated to 5 ml and mixed with 50 mL of ether. The resulting solid was collected by filtration and dried in vacuo. The product was dissolved in 25 mL of water, filtered and lyophilized to afford 0.246 g (89%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1yl-L-aspartic acid as a yellow-orange powder, m.p. 160° C. (dec).

HPLC: one peak on C18, k'=0.70, 85:15:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆)δ 13.12 (br s, COOH), 9.31 (s, 1H, NH₂), 9.08 (s, 1H, NH₂), 8.72 (s, 1H, pteridinyl 7-CH), 8.68 (br s, 1H, NH₂), 8.22 (d, 1H, J=8.0, amide NH), 8.09 (d, 1H, J=7.9, amide NH), 7.79 (br s, 1H, NH₂), 7.75 (d, 2H, J=8.9, aromatic CH), 6.82 (d, 2H, J=8.9, aromatic CH), 4.87 (s, 2H, pteridinyl-CH₂), 4.59-4.40 (m, 2H, methines), 3.24 (s, 3H, N--CH₃), 2.75-2.52 (m, 2H, asp CH₂), 2.27 (t, 2H, J=7.8, glu 4-CH₂), 2.10-1.78 (m, 2H, glu 3CH₂).

Elemental Analysis: Calcd. for C₂₄ H₂₇ N₉ O₈.2HCl1.3 H₂ O (MW 665.87): C, 43.29; H, 4.78; N, 18.93 ; Cl, 10.65. Found: C, 43.42; H, 4.90; N, 18.83; Cl, 10.46.

Mass Spectrum: (FAB) 570 (M+H)⁺.

UV Spectrum: (pH 7 buffer) λ_(max) (ε) 258.7 (21900), 306.4 (23800), 372.2 (7250). λ_(min) (ε) 240.1 (13400), 272.4 (15200), 345.0 (5740).

EXAMPLE 126 Di-tert-butyl N-((benzyloxy)carboxyl)-5-O-tert-butyl-L-glutam-1-yl-L-glutamate

According to example 9, 0.88 g of L-glutamic acid di-t-butyl ester hydrochloride (Sigma) was coupled with 1.00 g of N-Cbz-L-glutamic acid γ-t-butyl ester (Sigma) using 0.60 g of EDC (Aldrich) and 0.33 mL of N-methylmorpholine. The crude product was purified by flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 1.49 g (87%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-5-O-tert-butyl-L-glutamate as a thick transparent oil.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 8.21 (d, 1H, J=7.6, NH), 7.42 (d, 1H, J=8.2, NH), 7.33 (m, 5H, aromatic CH), 5.01 (m, 2H, PhCH₂ O), 4.17-3.96 (m, 2H, methines), 2.24 (m, 4H, glu 4-CH₂), 1.96-1.64 (m, 4H, glu 3-CH₂), 1.37 (s, 27H, t-Bu).

EXAMPLE 127 Di-tert-butyl 5-O-tert-butyl-L-glutam-1-yl-L-glutamate

According to example 48, 0.60 g of tert-butyl N-((benzyloxy)carboxyl)-5-O-tert-butyl-L-glutam-1-yl-5-O-tert-butyl-L-glutamate was hydrogenolyzed using 0.10 g of 10% Pd(C) in 45 mL of MeOH. This afforded 0.45 g (98%) of di-tert-butyl 5-O-tert-butyl-L-glutam-1-yl-L-glutamate as a thick transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 10 mg of the product with excess 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.86 (d, 1H, J=7.3, NH), 8.31 (br s, 3H, NH₃ ⁺), 4.21 (m, 1H, methine), 3.87 (m, 1H, methine), 2.35 (m, 4H, glu 4-CH₂), 2.07-1.66 (m, 4H, glu 3-CH₂), 1.39 (s, 27H, t-Bu).

EXAMPLE 128 Di-tert-butyl- 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamate

According to example 49, 0.30 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich was coupled with 0.386 g of di-tert-butyl 5-O-tert-butyl-L-glutam-1-yl-L-glutamate using 0.36 mL of DECP (Aldrich) and 0.39 mL of Et₃ N in 25 mL of DMF. The crude product was subjected to flash chromatography on 150 g of silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.432 g (73%) of di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamate as a yellow powder, m.p. 130-145° C.

EXAMPLE 129 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamic acid (Procedure B)

For Procedure A to prepare this compound, see Example 39.

According to example 50, 0.382 g of di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamate was treated with gaseous HCl in 50 mL of CH₃ NO₂ for 1 h. The reaction mixture was concentrated to 10 mL and mixed with 50 mL of ether. The resulting solid was collected by filtration and dried in vacuo. The product was dissolved in 25 mL of water, filtered and lyophilized to afford 0.265 g (76%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-glutamic acid as a yellow-orange powder, m.p. 155° C. (dec).

HPLC: one peak on C18, k'=0.88, 85:15:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆), δ 13.18 (br s, COOH), 9.29 (s, 1H, NH₂), 9.07 (s, 1H, NH₂), 8.72 (s, 1H, pteridinyl 7-CH), 8.69 (br s, 1H, NH₂), 8.19 (d, 1H, J=7.7, amide NH), 8.09 (d, 1H, J=7.9, amide NH), 7.87 (br s, 1H, NH₂), 7.74 (d, 2H, J=8.9, aromatic CH), 6.81 (d, 2H, J=8.9, aromatic CH), 4.87 (s, 2H, pteridinyl-CH₂), 4.42 (m, 1H, methine), 4.19 (m, 1H, methine), 3.24 (s, 3H, N--CH₃), 2.28 (m, 4H, glu 4-CH₂), 2.07-1.71 (m, 4H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₂₅ H₂₉ N₉ O₈ .1.9 HCl 2 H₂ O (MW 688.87): C, 43.59, H, 5.11; N, 18.30; Cl, 9.78 . Found: C, 43.72; H, 5.07; N, 18.16; Cl, 9.97.

Mass Spectrum: (FAB) 584 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 258.8(23700), 306.2 (25600), 371.9 (7830). λ_(min) (ε) 240.4 (14300), 272.9 (16600), 344.6 (6150).

EXAMPLE 130 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-phenylalanine (Procedure B)

For Procedure A to prepare this compound, see Example 97.

According to example 50, 3.51 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-phenylalaninate was treated with gaseous HCl in 150 mL of CH₃ NO₂ for 1 h. The reaction mixture was concentrated in vacuo to a slurry and mixed with 100 mL of ether. The solid was collected by vaccum filtration and dissolved in 150 mL of 6:4 MeCN:H₂ O. The solution was subjected to rotary evaporation to remove MeCN and the resulting suspension was frozen and lyophilized to afford 3.167 g (93%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-phenylalanine as a yellow-orange powder, m.p. 165° C. (dec).

HPLC: one peak on C18, k'=2.30, 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 ml/min. ¹ H-NMR: (300 MHz, DMSO-d₆)δ 13.35-12.95 (br s, COOH), 9.30 (s, 1H, NH₂), 9.07 (s, 1H, NH₂), 8.71 (s, 1H, pteridinyl 7-CH), 8.68 (br s, 1H, NH₂), 8.14 (d, 1H, J=7.6, amide NH), 8.02 (d, 1H, J=7.8, amide NH), 7.89 (br s, 1H, NH₂), 7.71 (d, 2H, J=8.9, aromatic CH), 7.21 (s, 1H, aromatic CH), 7.18-7.05 (m, 2H, aromatic CH), 6.98 (d, 1H, J=7.4, aromatic CH), 6.79 (d, 2H, J=8.9, aromatic CH), 4.85 (s, 2H, pteridinyl-CH₂), 4.39 (m, 2H, methines), 3.23 (s, 3H, N--CH₃), 3.03 (m, 1H, ArCH₂), 2.90 (m, 1H, ArCH₂), 2.20 (t, 2H, J=7.6, glu 4-CH₂), 2.01-1.76 (m, 2H, glu 3-CH₂), 1.19 (s, 9H, t-Bu).

Elemental Analysis: Calcd. for C₃₃ H₃₉ N₉ O₆ 1.5HCl 1.8 H₂ O (MW 744.85): C, 53.21; H, 5.97; Cl, 7.14. Found: C, 53.20; H, 5.97; N, 16.96; Cl, 7.20.

Mass Spectrum: (Ion Spray) 658 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 259.5(24400), 308.0 (24400), 371.5 (7410). λ_(min) (ε) 241.4 (14600), 274.2 (16200), 345.1 (5680).

EXAMPLE 131 tert-Butyl 5-O-tert-butyl-N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

A dry 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.150 g of 4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorbenzoic acid, 0.198 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate, and 7 mL of anhydrous DMF. The mixture was cooled to 0° C. and treated with 0.058 mL of Et₃ N followed by 0.063 mL of DECP. The solid starting material rapidly dissolved to give a clear solution. The solution was allowed to warm to RT and was stirred at RT under nitrogen. After 3 h, tlc (SiO₂, 95:5 CH₂ Cl₂ :MeOH) indicated a major new component at R_(F) =0.41 and minor components at R_(f) =0.38 and 0.06. The solution was concentrated in vacuo to dryness and the residue was dissolved in 70 mL of CHCl₃. The CHCl₃ solution was washed with 5% aqueous NaHCO₃ (3×60 mL), dried over anhydrous Na₂ SO₄ and concentrated to give a yellow glass. The crude product was purified by flash chromatography on silica gel (95:5→93:7 CH₂ Cl₂ :MeOH) to afford 0.258 g (80%) of tert-butyl 5-O-tert-butyl-N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a transparent glass.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 12.54 (s, 1H, benzoquinazoline NH), 9.86 (s, 1H, aromatic CH), 8.39 (d, 1H, J=7.6, amide NH), 8.23 (d, 1H, J=9.0, aromatic CH), 8.02 (d, 1H, J=8.4, aromatic CH), 7.68-7.41 (m, 4H, aromatic CH, amide NH), 7.35 (m, 1H, 4-aminobenzoyl NH), 7.21-6.97 (m, 4H, aromatic CH), 6.56 (d, 1H, J=8.8, aromatic CH), 6.41 (d, 1H, J=15.0, aromatic CH), 4.67-4.30 (m, 4H, benzoquinazoline 9-CH₂, methines), 3.06-2.77 (m, 3H, ArCh₂, cyclopentyl methine), 2.43 (s, 3H, CH₃), 2.19 (m, 2H, glu 4-CH₂), 2.00-1.38 (m, 10H, glu 3-CH₂, cyclopentyl CH₂), 1.35 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu).

EXAMPLE 132 N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine

According to example 50, 0.25 g of tert-butyl 5-O-tert-butyl-N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-1-yl-3cyclopentyl-L-phenylalaninate was treated with gaseous HCl in 40 mL of CH₃ NO₂ for 1 h. The reaction mixture was concentrated in vacuo to give a yellow residue. Analysis of this material by HPLC (C18, 65:35:0.1 H₂ O:MeCN:TFA) indicated a major component at k'=2.82 and minor components at k'=3.33, 1.53, and 0.83. The crude product ws subjected to semi-preparative reverse phase HPLC (C18, 65:35:0.1 H₂ O:MeCN:TFA). Fractions containing pure product (as determined by analytical HPLC) were combined and concentrated to dryness. The residue was mixed with 20 ml of water and treated with sufficient 1N aqueous NaOH to give complete solution. The solution was filtered and acidified to pH 3.0 by addition of 1N aqueous HCl. A white precipitate resulted which was separated by centrifugation, washed with 4 cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.141 g (62%) of N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-2-fluorobenzoyl)-L-glutam-1yl-3-cyclopentyl-L-phenylalanine as a fluffy white powder, m.p>200° C.

HPLC: one peak on C18, k'=2.77, 65:55:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆)δ 12.90-11.80 (br, m COOH, benzoquinazoline NH), 9.82 (s, 1H, aromatic CH), 8.27 (d, 1H, J=7.9, amide NH), 8.19 (d, 1H, J=8.7, aromatic CH), 7.99 (d, 1H, J=8.4, aromatic CH), 7.58 (m, 2H, aromatic CH), 7.45 (m, 2H, aromatic CH, amide NH), 7.31 (m, 1H, 4-aminobenzoyl NH), 7.05 (m, 2H, aromatic CH), 6.97 (d, 2H, J=7.9, aromatic CH), 6.51 (d, 1H, J=8.7, aromatic CH), 6.37 (d, 1H, J=15.2, aromatic CH), 4.55 (d, 2H, J=5.2, benzoquinazoline 9-CH₂), 4.43 (m, 2H, methines, 3.02 (m, 1H, ArCH₂), 2.82 (m, 2H, ArCH₂, cyclopentyl methine), 2.41 (s, 3H, CH₃), 2.16 (t, 2H, J=7.7, glu 4-CH₂), 1.98-1.71 (m, 4H, glu 3-CH₂, cyclopentyl CH₂), 1.69-1.31 (m, 6H, cyclopentyl CH₂).

Elemental Analysis: Calcd. for C₄₀ H₄₀ N₅ O₇ F 0.5H₂ O (MW 730.79): C, 65.74; H, 5.65; N, 9.58. Found: C, 65.76; H, 5.68; N, 9.57.

Mass Spectrum: (FAB) 722 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 265.9 (47900), 299.6 (27300), 348.4 (5710). λ_(min) (ε) 240.6 (19900), 284.5 (24900), 340.0 (3450). sh (e) 332.3 (6780).

EXAMPLE 133 tert-Butyl N-(4-(((2-amino-3,4-dihydro-4--oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate

A 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.097 g of 4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoic acid (Jones, T. R., et al, J. Med. Chem., 1986, 29, 1114; Nair, M. G., et al, J. Med. Chem., 1986, 29, 1754; Ghazala, M., et al, J. Med. Chem., 1986, 29, 1263; Acharya, S. P., et al, J. Heterocyclic Chem., 1975, 12, 1283), 0.142 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate, 0.044 g of 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (Aldrich, and 7 mL of anhydrous DMF. The mixture was treated with 0.057 g of EDC and was stirred at RT under nitrogen. The solid starting material slowly dissolved to give a clear solution. After 3 days the solution was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on silica gel (98.2→95:5 CH₂ Cl₂ :MeOH) to afford 0.142 g (65%) of tert-butyl N-(4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate as a white solid, m.p. 115° C. (dec).

EXAMPLE 134 N-(4(((2-Amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine

According to example 50, 0.114 g of tert-butyl N-(4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-phenylalaninate was treated with gaseous HCl in CH₃ NO₂ for 2 h. The reaction mixture was concentrated to dryness and the residue was suspended in 50 mL of ether. The resulting solid was collected by filtration and dried in vacuo. The crude product was purified by semi-preparative reverse phase HPLC (C18, 68:32:0.1→60:40:0.1 H₂ O:MeCN:TFA). Fractions containing pure product (as determined by analytical HPLC) were combined and concentrated to dryness. The residue was suspended in water and lyophilized to afford 0.053 g (45%) of N-(4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoyl)-L-glutam-1-yl-3- cyclopentyl-L-phenylalanine as a fluffy white solid, m.p. 158° C. (dec).

HPLC: two peaks on C18;k'=1.27 (98.8%), k'=0.50 (1.2%); 62:38:0.1 H₂ O:MeCN:TFA; flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 12.90-11.80 (br m, COOH), 8.07 (d, 1H, J=7.9, amide NH), 8.03 (d, 1H, J=7.9, amide NH), 7.87 (s, 1H, aromatic CH), 7.71 (d, 2H, J=8.7, aromatic CH), 7.62 (d, 1H, J=8.6, aromatic CH), 7.57-7.24 (br s, 2H, NH₂), 7.31 (d, 1H, J=8.6, aromatic CH), 7.13-6.95 (m, 4H, aromatic CH), 6.82 (d, 2H, J=8.8, aromatic CH), 4.71 (s, 2H, quinazolinyl-CH₂), 4.41 (m, 2H, methines), 4.30 (s, 2H, propargyl CH₂), 3.19 (s, 1H, propargyl CH), 3.10-2.77 (m, 3H, ArCH₂, cyclopentyl methine), 2.23 (t, 2H, J=7.6, glu 4-CH₂), 2.02-1.76 (m, 4H, glu 3-CH₂, cyclopentyl CH₂, 1.75-1.38 (m, 6H, cyclopentyl CH₂).

¹⁹ F-NMR: (282 MHz, DMSO-d₆)δ 1.53 (relative to TFA external standard).

Elemental Analysis: Calcd. for C₃₈ H₄₀ N₆ O₇ TFA 2.2H₂ O (MW 846.43): C, 56.76; H, 5.41; N, 9.93. Found: C, 56.79; H, 5.42; N, 9.95.

Mass Spectrum: (FAB) 693 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 227.2 (48400), 306.4 (24500). λ_(min) (ε) 213.5 (40200), 252.2 (11500).

EXAMPLE 135 Ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-L-phenylalaninate

A 500 mL round bottom flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 10.0 g of N-benzyloxycarbonyl-L-glutamic acid γ-ethyl ester, 7.43 g of L-phenylalanine ethyl ester hydrochloride (Aldrich), 100 mL of CH₂ Cl₂, and 4.51 mL of Et₃ N. The solution was cooled to 0° C. and treated with a solution of 6.67 g of DCC (Fluka) in 25 ml of CH₂ Cl₂. The solution was stirred at 0° C. for 1 h and then allowed to warm to RT. After an additional 3 h, the mixture was filtered to remove dicyclohexylurea. The filtrate was washed with 10% aqueous citric acid (3×50 mL), saturated aqueous NaHCO₃ (3×50 mL), followed by one 60 mL portion of water. The solution was dried over anhydrous Na₂ SO₄, concentrated to 30 mL, and triturated with addition of 50 mL of ether followed by 150 mL of pentane. The resulting precipitate was collected by filtration and dried in vacuo to afford 12.9 g (82%) of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-L-phenylalaninate as a white crystalline solid, m.p. 85-87° C.

EXAMPLE 136 Ethyl 5-O-ethyl-L-glutam-1-yl-L-phenylalaninate

According to example 10, 13.08 g of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-L-phenylalaninate was hydrogenolyzed in 300 mL of EtOH using 2.43 g of 10% Pd(C) and 2 mL of acetyl chloride to afford 9.84 g (94%) of ethyl 5-O-ethyl-L-glutam-1-yl-L-phenylalaninate HCl as a white foam.

¹ H-NMR: (200 MHz, DMSO-d₆)δ 9.08 (d, 1H, J=7.0, NH), 7.90-7.40 (br s, 3H, NH3⁺), 7.29 (m, 5H, aromatic CH), 4.50 (m, 1H, methine), 4.07 (m, 4H, ethyl CH₂), 3.80 (m, 1H, methine), 3.04 (m, 2H, ArCH₂), 2.46 (m, 2H, glu 4-CH₂), 1.99 (m, 2H, glu 3-CH₂), 1.20 (t, 3H, J=7.1, ethyl CH₃), 1.13 (t, 3H, J=7.1, ethyl CH₃).

EXAMPLE 137 Ethyl N-((S)-4-(ethoxycarbonyl)-2-(4-nitrophthalimido)butanoyl)-L-phenylalaninate

A 1 L round bottom flask equipped with a magnetic stirrer, a condenser, a nitrogen inlet, and a Dean Stark trap was charged with 7.28 g of ethyl 5-O-ethyl-L-glutam-1-yl-L-phenylalaninate HCl, 3.68 g of 4-nitrophthalic anhydride (American Tokyo Kasei, Portland, Oreg. 97203), 300 mL of toluene, and 2.43 g of diisopropylethylamine (Aldrich). The reaction mixture was heated at reflux for 2 h, allowed to cool to RT, and stirred at RT overnight under nitrogen. The toluene was removed by rotary evaporation and the residue was dissolved in 400 mL of CH₂ Cl₂. The solution was washed with water (2×200 mL), 5% aqueous NaHCO₃ (2×250 mL), and dried over anhydrous MgSO₄. The drying agent was removed by filtration and the filtrate was concentrated to 100 mL. The concentrate was mixed with excess ether to induce precipitation. The precipitate was collected by filtration and dried in vacuo to afford 6.72 g (67%) of ethyl N-((S)-4-(ethoxycarbonyl)-2-(4-nitrophthalimido)butanoyl)-L-phenylalaninate as a white solid. ¹ H-NMR: (300 MHz, DMSO-d₆)δ 8.65 (m, 2H, aromatic CH, NH), 8.49 (s,1H, aromatic CH), 8.13 (d, 1H, J=8.6, aromatic CH), 7.27-7.06 (m, 5H, aromatic CH), 4.70 (m, 1H, methine), 4.39 (m, 1H, methine), 4.04 (q, 2H, J=7.1, ethyl CH₂), 3.93 (q, 2H, J=7.1, ethyl CH₂), 2.94 (dd; 1H: J=13.7, 5.8; ArCH₂) ,2.82 (dd; 1H; J=13.6, 9.4, ArCH₂), 2.34 (m, 3H, glu 4-CH₂, glu 3-CH₂), 2.18 (m, 1H, glu 3-CH₂), 1.11 (t, 6H, J=7.1, ethyl CH₃).

EXAMPLE 138 Ethyl N-((S)-2-(4-aminophthalimido)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate

To a 300 mL Parr bottle were added 1.17 g of 10% Pd(C) and 20 mL of EtOAc under a nitrogen flush. To this mixture was added a solution of 6.72 g of ethyl N-((S)-4-(ethoxycarbonyl)-2-(4-nitrophthalimido)butanoyl)-L-phenylalaninate in 200 mL of EtOAc. The mixture was deoxygenated by bubbling nitrogen through and then hydrogenated at 45 psi for 1 h. The bottle was purged with nitrogen and the catalyst was removed by filtration through celite. The filtrate was concentrated in vacuo to give a residue which was purified by flash chromatography or silica gel (1:1→3:7 hexane:EtOAc). This afforded 5.66 g (88%) of ethyl N-((S)-2-(4-aminophthalimido)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate as a bright yellow solid.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 8.50 (d, 1H, J=7.4, NH), 7.48 (d, 1H, J=8.2, aromatic CH), 7.26-7.08 (m, 5H, aromatic CH), 6.92 (s, 1H, aromatic CH), 6.82 (dd; 1H; J=8.3, 1.6; aromatic CH), 6.47 (s, 2H, NH₂), 4.54 (m, 1H, methine), 4.35 (q, 1H, J=7.4, methine), 4.07-3.86 (m, 4H, ethyl CH₂), 2.92 (m, 2H, ArCH₂), 2.40-2.13 (m, 4H, glu 4-CH₂, glu 3-CH₂), 1.09 (m, 6H, ethyl CH₃).

EXAMPLE 139 Ethyl N-((S)-2-(5-amino-1-oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate

A zinc-mercury amalgam was prepared by stirring 19.8 g of zinc dust (Mallinckrodt, Inc., Paris, Ky., 40361) in 500 mL of 10% aqueous HCl for 10 min. The HCl solution was decanted and the zinc was washed with water until neutral pH was achieved. A warm solution of 0.10 g of HgCl₂ in water was added to the zinc and the mxture was stirred for 10 min. The amalgam was collected by filtration, washed consecutively with with water, EtOH, and ether and air dried. The dry amalgam was added to a solution of 3.0 g of ethyl N-((S)-2-(4-aminophthalimido)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate and 60 mL of 1N ethereal HCl in 250 mL of EtOH which was maintained at 0° C. in an ice bath. After stirring at 0° C. for 25 min, tlc (SiO₂, 3:7 hexane:EtOAc) indicated only a trace of starting material at Rf=0.58 and two new components at Rf=0.52 and 0.63. The reaction mixture was filtered to remove the amalgam and the filtrate was concentrated in vacuo to 200 mL. The solution was hydrogenated at 50 psi in the presence of 0.54 g of 10% Pd(C) for 18 h. The catalyst was removed by filtration through celite and the filtrate was neutralized by addition of excess NaHCO₃ and then concentrated to dryness. The residue was partitioned between water and CH₂ Cl₂ and the layers were separated. The CH₂ Cl₂ solution was washed with water (2×200 mL) and the water layer was extracted with EtOAc (200 mL). The CH₂ Cl₂ and EtOAc solutions were combined, dried over anhydrous Na₂ SO₄, and concentrated to give 2.77 g of a pale yellow foam. Analysis of this material by tlc (SiO₂, 9:1 CH₂ Cl₂ :acetone) indicated four major components at R_(f) =0.50, 0.41 0.29, and 0.20. These materials were separated by flash chromatography on silica gel (9:1 CH₂ Cl₂ :acetone). Comparative ¹ H-NMR analysis indicated the R_(f) =0.29 material to be ethyl N-((S)-2-(5-amino-1-oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate. The product was obtained as a pale yellow foam, 0.646 g (22%). ¹ H-NMR: (300 MHz, DMMSO-d₆),δ 8.48 (d, 1H, J=7.7, NH), 7.32 (d, 1H, J=8.2, aromatic CH), 7.14 (s, 5H, aromatic CH), 6.59 (m, 2H, aromatic CH), 5.78 (s, 2H, NH₂), 4.73 (m, 1H, methine), 4.43 (m, 1H, methine), 4.13-3.90 (m, 6H, ethyl CH₂, isoindolinyl methylene), 3.07-2.85 (m, 2H, ArCH₂), 2.26-2.01 (m, 3H, glu 4-CH₂, glu 3-CH₂), 1.88 (m, 1H, glu 3-CH₂), 1.12 (m, 6H, ethyl CH₃).

EXAMPLE 140 Ethyl N-((S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate

According to example 5, 1.04 g of 9-bromomethyl-3-methylbenzo(f)quinazolin-1-(2H)-one was reacted with 1.65 g of ethyl N-((S)-2-(5-amino-1-oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate using 0.84 g of NaHCO₃. The reaction mixture was stirred at 100° C. for 5 h, cooled to RT, and stirred at RT for 18 h. Analysis by tlc (SiO₂, 4:1 CH₂ Cl₂ :acetone) indicated incomplete reaction. An additional 0.3 g of 9-bromomethyl-3-methylbenzo(f)quinazolin-1(2H)-one was added and the reaction mixture was stirred at 100° C. for 4 h. The mixture was cooled to RT and filtered to remove solids. The crude product was absorbed onto 25 g of silica gel by adding silica gel to the filtrate, concentrating to dryness, and storing in vacuo. The product/silica gel mixture was loaded onto a column (1000 g SiO₂) and subjected to flash chromatography (CH₂ Cl₂ →95:5 CH₂ Cl₂ :MeOH) to give 0.30 g of pure product. Impure chromatography fractions were combined, concentrated, and subjected to flash chromatography a second time (98:2→95:5 CH₂ Cl₂ :MeOH) to give 0.26 g of pure product. This afforded a total yield of 0.56 g (23%) of ethyl N-((S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate as a white solid, m.p. 140° C. (dec).

EXAMPLE 141 N-((S)-4-Carboxy-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)butanoyl)-L-phenylalanine

To a 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.90 g of ethyl N-((S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate, 3 mL of EtOH and 10 mL of 0.2N aqueous NaOH. The solution was stirred at RT under nitrogen for 2 h and was acidified to pH 3.0 by addition of 1N HCl. A white precipitate resulted which was separated by centrifugation. Analysis of this material by analytical HPLC (C18, 70:30:0.10 H₂ O:MeCN:TFA) indicated a major component at K'=1.93 (90%) and a minor component at k'=2.41. The crude product was purified by semi-preparative reverse phase HPLC (C18, 75:25:0.1 H₂ O:MeCN:TFA). Fractions containing pure product (k'=1.93 by analytical HPLC using above conditions) were combined and concentrated in vacuo to dryness. The residue was dissolved in 15 mL of 0.1N aqueous NaOH and the solution was adjusted to pH 3.0 by addition of 1N HCl. The resulting precipitate was collected by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.035 g (40%) of N-((S)-4-carboxy-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)butanoyl)-L-phenylalanine as a white powder, m.p. 180° C.

HPLC: one peak on C18, k'=2.3, 70:30:0.10 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆)δ 12.83-12.56 (br s, 1H, COOH), 12.73 (s, 1H, benzoquinazoline NH), 12.20-11.90 (br s, 1H, COOH), 9.88 (s, 1H, aromatic CH), 8.19 (m 2H, amide NH, aromatic CH), 8.00 (d, 1H, J=8.4, aromatic CH), 7.66 (d, 1H, J=7.4, aromatic CH), 7.57 (d, 1H, J=8.7, aromatic CH), 7.33 (d, 1H, J=8.6, aromatic CH), 7.17 (m, 1H, isoindolinyl 5-NH), 7.03 (d, 2H, J=7.2, aromatic CH), 6.90 (m, 3H, aromatic CH), 6.72 (d 1H, J=7.8, aromatic CH), 6.59 (s, 1H, aromatic CH), 4.67 (m, 1H, methine), 4.57 (d, 2H, J=5.9, benzoquinazoline 9-CH₂), 4.38 (m, 1H, methine), 4.02 (d, 1H, J=16.8, isoindolinyl methylene), 3.76 (d, 1H, J=16.9, isoindolinyl methylene), 3.00 (m, 1H, ArCH₂), 2.80 (m, 1H, ArCH₂), 2.41 (s, 3H, CH₃), 2.02 (m, 3H, glu 4-CH₂, glu 3-CH₂), 1.76 (m, 1H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₃₆ H₃₃ N₅ O₇ 2.3H₂ O (MW 689.12): C, 62.75; H, 5.50; N, 10.16. Found: C, 62.82; H, 5.50; N, 10.18.

Mass Spectrum: (FAB) 648 (M+H) ⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 265.6 (38900), 301.7 (21100), 348.4 (4870). λ_(min) (ε) 245.1 (19700), 284.1 (18300), 341.8 (3420). sh (e) 331.7 (7570).

EXAMPLE 142 tert-Butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(trimethylsilyl)benzyl)-4-morpholinecarboxylate

A dry 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 2.08 g of t-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich), 1.43 g of 3-(trimethylsilyl)benzyl bromide (Yamakawa, T., et al, J. Med. Chem., 1990, 33, 1430), and 45 mL of anhydrous THF. The solution was cooled to -78° C. and was treated with 6.17 mL of 1 M sodium bis(trimethylsilyl)amide/THF (Aldrich) via syringe through a rubber septum. The solution was stirred at -78° C. for 3.5 h and was then poured into 150 mL of water. The resulting mixture was extracted with EtOAc (4×50 mL). The combined EtOAc extracts were washed with saturated brine (3×100 mL), dried over anhydrous Na₂ SO₄, and concentrated in vacuo to give a light yellow oil. The crude material was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 2.6 g (88%) of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(trimethylsilyl)benzyl)-4-morpholinecarboxylate as a white powder, m.p. 88-90° C.

EXAMPLE 143 (2R, 3S, 5S)-6-Oxo-2,3-diphenyl-5-(3-(trimethylsilyl)benzyl)morpholine

According to example 82, 2.47 g of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(trimethylsilyl)benzyl)-4-morpholinecarboxylate was treated with 4.1 mL of TFA in 40 mL of CH₂ Cl₂ for 3 h. Neutralization with 8 mL of Et₃ N and work-up in the usual manner gave a thick oil. Purification of the crude material by flash chromatography on 80 g of silica gel (85:15 hexane:EtOAc) afforded 1.70 g (83%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(trimethylsilyl)benzyl)benzyl)morpholine as a white crystalline solid, m.p. 94-95° C.

EXAMPLE 144 tert-Butyl 3-(trimethylsilyl)-L-phenylalaninate

According to example 53, 158 g of 2R, 3S 5S)-6-oxo-2,3-diphenyl-5-(3-(trimethylsilyl)benzyl)morpholine was hydrogenolyzed in 70 mL of 1:1 THF:EtOH using 0.70 g of PdCl₂. Analysis of the product ¹ H-NMR indicated partial desilylation of the phenyl ring. The mixture was subjected to esterification according to example 54 using 25 mL of isobutylene and 0.5 mL of conc. H₂ SO₄ in 25 mL of 1,4-dioxane. Work-up in the usual manner afforded a light yellow oil which by tlc (SiO₂, EtOAc) consisted of two components at R_(f) =0.53 and 0.47. The R_(f) =0.53 material was isolated by flash chromatography twice on silica gel (EtOAc then 1:1 hexane:EtOAc). This gave 0.161 g (15%) of tert-butyl 3-(trimethylsilyl)-L-phenylalaninate as a clear oil. A sample of the corresponding HCl salt was prepared for ¹ H-NMR by treating 5 mg of product with excess 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (200 MHz, DMSO-d₆)δ 8.53 (br s, 3H, NH₃ ⁺), 7.50-7.20 (m, 4H, aromatic CH), 4.14 (m, 1H, methine), 3.21 (m, 1H, ArCH₂), 2.98 (m, 1H, ArCH₂), 1.26 (s, 9H, t-Bu), 0.23 (s, 9H, trimethylsilyl).

EXAMPLE 145 tert-Burtyl N-(benzyloxy)carbonyl)-5-O-tert-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate

To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.161 g of tert-butyl 3-(trimethlsilyl)-L-phenylalaninate, 0.194 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma), and 10 mL of CH₂ Cl₂. The solution was cooled in to 0° C. and was treated with 0.110 g of EDC. The reation mixture was stirred of 0° C. for 1.5 h and was then allowed to warm to RT. After an additional 16 h of stirring at RT, the solution was concentrated in vacuo to dryness in the presence of 5 g of silica gel. The resulting mixture was applied to a column and the product isolated by flash chromatography (SiO₂, 75:25 hexane:EtOAc) to afford 0.320 g (95%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1yl-3-(trimethylsilyl)-L-phenylalaninate as a thick, transparent glass.

¹ H-NMR: (200 MHz, DMSO-d₆)δ 8.29 (d, 1H, J=7.0, NH), 7.50-7.18 (m, 10H, aromatic CH, NH), 5.02 (s, 2H, PhCH₂ O), 4.38 (m, 1H, methine), 4.07 (m, 1H, methine), 2.97 (d, 2H, J=7.3, ArCH₂), 2.23 (t, 2H, J=7.6, glu 4-CH₂), 1.96-1.60 (m, 2H, glu 3-CH₂), 1.40 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu), 0.24 (s, 9H, trimethylsilyl).

EXAMPLE 146 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate

According to example 48, 0.315 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate was hydrogenolyzed using 0.051 g of 10% Pd(C) in 35 mL of MeOH. This afforded 0.24 g (98%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 5 mg of the product with excess 1N ethereal HCl and concentrating to dryness. ¹ H-NMR: (200 MHz, DMSO-d₆)δ 8.99 (d, 1H, J=7.2, NH), 8.28 (br s, 3H, NH₃ ⁺), 7.47-7.24 (m, 4H, aromatic CH), 4.42 (m, 1H, methine), 3.86 (m, 1H, methine), 3.01 (d, 2H, J=7.2, ArCH₂), 2.37 (m, 2H, glu 4-CH₂), 2.01 (m, 2H, glu 3-CH₂), 1.42 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu), 0.26 (s, 9H, trimethylsilyl).

EXAMPLE 147 tert-Butyl N-(4-(((2,4-diamino-6-pyeridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate

According to example 49, 0.095 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.12 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate using 0.11 mL of DECP (Aldrich) and 0.12 mL of Et₃ N in 11 mL of DMF. The crude product was subjected to flash chromatography on silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.141 g (72%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate as a yellow powder, m.p. 188-125° C.

¹ H-NHR: (200 MHz, DMSO-d₆)δ 8.58 (s, 1H, pteridinyl 7-CH), 8.23 (d, 1H, J=7.5, amide NH), 8.00 (d, 1H, J=7.8, amide NH), 7.74 (d, 2H, J=8.7, aromatic CH), 7.70 (br s, 1H, NH₂), 7.48 (br s, 1H, NH₂), 7.33 (m, 2H, aromatic CH), 7.22 (m, 2H, aromatic CH), 6.83 (d, 2H, J=8.8, aromatic CH), 6.63 (br s, 2H, NH₂), 4.79 (s, 2H, pteridinyl-CH₂), 4.53-4.29 ((m, 2H, methines), 3.23 (s, 3H, N-CH₃), 2.96 (d, 2H, J=7.0, ArCH₂), 2.23 (m, 2H, glu 4-CH₂), 2.04-1.77 (m, 2H, glu 3-CH₂), 1.38 (s, 9H, t-Bu), 1.28 (s, 9H, t-Bu), 0.21 (s, 9, trimethylsilyl).

EXAMPLE 148 N-(4-(((2,4-Diamino-6-pyeridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalanine

A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.111 g of tert-butyl N-(4-(((2,4-diamino-6-pyeridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalaninate and 25 mL of CH₃ NO₂. The resulting solution was treated with 5 mL of TFA and stirred at RT. After 1 h, the solution was concentrated in vacuo to dryness. The residue was dissolved in 3 mL of CH₃ NO₂ and the solution was treated with 20 mL of ethyl ether. A yellow solid precipitated which was collected by filtration and dried in vacuo. The product was dissolved in 3 mL of 1:1 H₂ O:CH₃ CN and the solution mixed with 50 mL of water. A suspension resulted which was frozen and lyophilized to afford 0.90 g (82%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-(trimethylsilyl)-L-phenylalanine as a yellow powder, m.p. 155° C. (dec).

HPLC: one peak of C18, k'=1.55, 65:35:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min. ¹ H-NMR: (300 MHz, DMSO-d₆)δ 13.00-11.75 (br s, COOH), 8.64 (s, 1H, pteridinyl 7-CH), 8.60 (br s, 1H, NH₂), 8.39 (br s, 1H, NH₂), 8.11 (d, 1H, J=7.7, amide NH), 7.97 (d, 1H, J=8.1, amide NH), 7.75-7.10 (br m, 2H, NH₂), 7.69 (d, 2H, J=8.9, aromatic CH), 7.28 (m, 2H, aromatic CH), 7.14 (m, 2H, aromatic CH), 6.79 (d, 2H, J=8,9, aromatic CH), 4.82 (s, 2H, pteridinyl-CH₂), 4.39 (m, 2H, methines), 3.21 (s, 3H, N-CH₃), 3.02 (m, 1H, ArCH₂), 2.88 (m, 1H, ArCH₂), 2.20 (t, 2H, J=7.8, glu 4-CH₂), 2.00-1.72 (m, 2H, glu 3-CH₂), 0.17 (s, 9H, trimethylsilyl).

Elemental Analysis: Calcd. for C₃₂ H₃₉ N₉ O₆ Si.0.8 TFA.1.2 H₂ O (MW 786.64): C, 51.30; H, 5.41; N, 16.03. Found: C, 51.24; H, 5.39; N, 16.07.

Mass Spectrum: (FAB) 674 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 259.5 (23500), 308.0 (24300), 373.2 (7210). λ_(min) (ε) 240.7 (12700), 273.9 (15500), 345.0 (5210).

EXAMPLE 149 tert-Butyl 3-iodo-L-tyrosinate

A mixture of 5.0 g of 3-iodo-L-tyrosine (Aldrich) and 2.5 mL of conc. H₂ SO₄ in 85 mL of 1,4-dioxane was added to 30 mL of liquid isobutylene (condensed at -78° C.) in a 300 mL pressure bottle. The vessel was stoppered and the contents stirred at RT. After 3 days the bottle was cooled in an ice water bath, opened and the solution poured into a mixture of 10 g of NaHCO₃ in 150 mL of water. The resulting mixture was subjected to rotary evaporation to remove isobutylene and dioxane. A white solid resulted which was collected by filtration and dried in vacuo. Analysis by tlc (SiO₂, CH₂ Cl₂ :MeOH) indicated major and minor components at R_(f=) 0.52 and 0.60 respectively. The crude product was purified by flash chromatography on silica gel (95:5 CH₂ Cl₂ :MeOH) to afford 3.4 g (58%) of tert-butyl 3-iodo-L-tyrosinate as a white crystalline solid, m.p. 158-159° C.

EXAMPLE 150 tert-Butyl N-((benzyloxy)carbonyl)-4-O-((benzyloxy)carbonyl)-3-iodo-L-tyrosinate

A 500 mL round bottomed flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 5.15 g of tert-butyl 3-iodo-L-trosinate, 250 mL of 1,4-bioxane, and 4.55 mL of Et₃ N. The resulting solution was treated with 4.66 mL of benzyl chloroformate by slow addition. After 30 min, tlc (SiO₂, 8:2 hexane:EtOAc) indicated no remaining starting material and a single new component at R_(f) =0.50. The solution was concentrated in vacuo and the residue mixed with 200 mL of water. The mixture was extracted with EtOAc (4×50 mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO₃, dried over anhydrous MgSO₄, and concentrated in vacuo. The crude product was subjected to flash chromatography on silica gel (8:2 hexane:EtOAc) to afford 7.49 g (84%) of tert-butyl-N-((benzyloxy)carbonyl)-4-O-((benzyloxy)carbonyl)-3-iodo-L-tyrsinate as a transparent foam.

¹ H-NMR: (200 MHz, DMSO-d₆)δ 7.77 (m, 2H, NH, aromatic CH), 7.53-7.21 (m, 12H, aromatic CH), 5.32 (s, 2H, PhCH₂ O), 5.20 (s, 2H, PhCH₂ O), 4.12 (m, 1H, methine), 2.92 (m, 2H, ArCH₂), 1.35 (s, 9H, t-Bu).

EXAMPLE 151 tert-Butyl 3-cyclopentyl-L-tyrosinate

A 300 mL Parr bomb was charged with 5.13 g of tert-butyl N-((benzyloxy)carbonyl)-4-O-((benzyloxy)carbonyl)-3-iodo-L-tyrosinate, 120 mL of toluene, 30 mL of cyclopentene and 0.49 g of tri-ortho-tolylphosphine. The solution was deoxygenated by bubbling nitrogen through for 10 min and was treated with 0.182 g of Pd(OAc)₂ and 1.24 mL of Et3N. The bomb was flushed with nitrogen, sealed, and heated to 110° C. for 48 h. The vessel was cooled to RT, purged with nitrogen and opened. The reaction mixture was filtered to remove solids and the filtrate was concentrated in vacuo to dryness. The residue was dissolved in 200 mL of EtOAc and the solution was washed with water(4×100 mL), dried over anhydrous NaSO₄ and concentrated to give a thick brown oil. Analysis by tlc (SiO₂, 8:2 hexane:EtOAc) indicated two major components at R_(f) =0.50 and 0.40. The crude mixture was subjected to flash chromatography on silica gel (8:2 hexane:EtOAc). This afforded 2.0 g (43%) of the R_(f) =0.50 material identified as the bis(benzyloxycarbonyl) olefin mixture by ¹ H-NMR. In addition, 0.90 g (25%) of the R_(f) =0.40 material was obtained and was identified as the N-benzyloxycarbonyl olefin mixture resulting from loss of the O-benzyloxycarbonly group. The bis(benzyloxycarbonyl) olefin mixture (2.00 g) ws subjected to catalytic hydrogenation (45 psi) in the presence of 0.35 g of 10% Pd(C) in 70 mL of MeOH for 18 h. The crude product was purified by flash chromatography (SiO₂, EtOAc) to give 0.99 g (93%) of tert-butyl 3-cyclopentyl-L-tyrosinate as a transparent glass. The N-Benzyloxycarbonyl olefin mixture (0.90 g) was similarly hydrogenated and purified to afford 0.52 g (83%) of tert-butyl 3-cyclopentyl-L-tyrosinate.

¹ H-NMR: (200 MHz, DMSO-d₆)δ 9.05 (s, 1H, OH), 6.92 (d, 1H, J=1.9, aromatic CH), 6.80 (dd; 1H; J=8.0, 2.0; aromatic CH), 6.66 (d, 1H, J=8.0, aromatic CH), 3.16 (m, 1H, cyclopentyl methine), 2.67 (d, 2H, J=6.66, ArCH₂), 2.00-1.40 (m, 10H, cyclopentyl CH₂, NH₂), 1.33 (s, 9H, t-Bu).

EXAMPLE 152 tert-Butyl N-(benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate

According to example 145, 0.99 g of tert-butyl 3-cyclopentyl-L-tyrosinate was coupled with 1.09 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.65 g of EDC in 20 mL of CH₂ Cl₂ for 4 h. The crude product was purified by flash chromatography on silica gel (7:3 hexane:EtOAc) to afford 1.8 g (89%) of tert-butyl N-((benzyloxy)carboxyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyosinate as a colorless foam.

¹ H-NMR: (200 MHz, DMSO-d₆)δ 9.08 (s, 1H, OH), 8.17 (d, 1H, J=7.5, NH), 7.36 (m, 6H, aromatic CH, NH), 6.96 (s, 1H, aromatic CH), 6.81 (d, 1H, J=8.1, aromatic CH), 6.67 (d, 1H, J=8.1, aromatic CH), 5.01 (m, 2H, PhCH₂ O), 4.28 (m, 1H, methine), 4.08 (m, 1H, methine), 3.17 (m, 1H, cyclopentyl methine), 2.82 (d, 2H, J=6.9, ArCH₂), 2.21 (t, 2H, J=7.7, glu 4-CH₂), 1.99-1.46 (m, 10H, glu 3-CH₂, cyclopentyl CH₂), 1.40 (s, 9H, t-Bu), 1.33 (s, 9H, t-Bu).

EXAMPLE 153 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate

According to example 48, 1.75 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate was hydrogenolyzed using 0.28 g of 10% Pd(C) in 70 mL of MeOH for 3 h. The crude product was subjected to flash chromatography on silica gel (95:5 CH₂ Cl₂ :MeOH) to afford 1.26 g (92%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate as a white crysatalline solid, m.p. 108-110 C.

EXAMPLE 154 tert-Butyl N-(4(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate

to a dry 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 35 mL of anhydrous DMF, 1.14 mL of Et₃ N, and 0.99 mL of DECP. To the resulting solution were added 1.24 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich). The solid starting material slowly dissolved to give a yellow solution. After 45 min, a solution of 1.60 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate in 8 mL of DMF was added. The solution was stirred at RT for 24 h and was then concentrated in vacuo to dryness. The residue was dissolved in 150 mL of CHCl₃. The solution was washed with 5% aqueous NH₄ OH (3×70 mL), dried over anhydrous MgSO₄, and concentrated in vacuo. The crude product was subjected to flash chromatography on silica gel twice (7:7:1 CH₂ Cl₂ :acetone:MeOH, 93:7 CH₂ Cl₂ :MeOH) to afford 2.08 g (80%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate as a yellow powder, m.p. 145-148° C.

EXAMPLE 155 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-tyrosine

According to example 50, 2.04 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosinate was treated with gaseous HCl in 65 mL of CH₃ NO₂ for 1 h. The yellow suspension was concentrated to a slurry and mixed with 80 mL of ethyl ether. The resulting precipitate was collected by filtration and dried in vacuo. The product was dissolved in 40 mL of 8:2 H₂ O:MeCN and the solution was filtered and concentrated to 30 mL. A yellow suspension was obtained which was diluted with 150 mL if water, frozen, and lyophilized to give 1.71 g (86%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-tyrosine as a yellow-orange powder, m.p. 175° C. (dec).

HPLC: one peak on C18, k'=2.98, 75:25:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.12 (br m, COOH), 9.30 (s, 1H, NH₂), 9.09 (s, 1H, NH₂), 8.71 (s, 1H, pteridinyl 7-CH), 8.67 (br s, 1H, NH₂), 8.02 (m, 2H, amide NH's), 7.78 (br s, 1H, NH₂), 7.71 (d, 2H, J=8.7, aromatic CH), 6.91 (s, 1H, aromatic CH), 6.79 (m, 3H, aromatic CH), 6.61 (d, 1H, J=8.1, aromatic CH), 4.86 (s, 2H, pteridinyl-CH₂), 4.42 (m, 1H, methine), 4.29 (m, 1H, methine), 3.24 (s, 3H, N--CH₃), 3.09 (m, 1H, cyclopentyl methine), 2.93-2.71 (m, 2H, ArCH₂), 2.23 (t, 2H, J=7.8, glu 4-CH₂), 2.03-1.74 (m, 4H, glu 3-CH₂, cyclopentyl CH₂), 1.73-1.37 (m, 6H, cyclopentyl CH₂).

Elemental Analysis: Calcd. for C₃₄ H₃₉ N₉ O₇.1.6HCl.1.7H₂ O (MW 774.70): C, 52.71; H, 5.72; N, 16.27; Cl, 7.32. Found: C, 52.79; H, 5.71; N, 16.16; Cl, 7.35.

Mass Spectrum: (Ion Spray) 686 (M+H)⁺.

UV Spectrum: (pH 7 Buffer λ_(max) (ε) 259.3 (24700), 307.8 (24800), 372.5 (7780). λ_(min) (ε) 242.3 (15500), 272.9 (17800), 344.6 (6080). sh(e) 221.3 (28900), 286.4 (21100).

EXAMPLE 156 3-(3-hydroxy-3-pentyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol

To a dry 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 5.0 g of 3-iodo-O-(tert-butyldimethylsilyl)benzyl alcohol and 40 mL of anhydrous ethyl ether. The solution was cooled to -78° C. under nitrogen and was treated with 23.2 mL of 1.3 M sec-butyllithium/cyclohexane (Aldrich) via syringe. After 20 min, the solution was treated with 3.1 mL of 3-pentanone (Aldrich). The reaction mixture was stirred at -78° C. for 1.5 h and was then allowed to warm to RT. After 3 h at RT the solution was quenched by addition of 5 mL of water, stirred for several minutes and then mixed with 80 mL of water. The two phases were separated and the aqueous solution was extracted with an additional 50 mL portion of ether. The ether extract was combined with the original ether solution. This solution was washed with saturated aqueous brine (3×50 mL), dried over anhydrous MgSO₄, and concentrated to afford a clear liquid. Analysis by tlc (SiO₂, 9:1 hexane:EtOAc) indicated a major new component at R_(f) =0.47. The crude product was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 3.9 g (88%) of 3-(3-hydroxy-3-pentyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol as a clear liquid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.33 (s, 1H, aromatic CH), 7.26 (m, 2H, aromatic CH), 7.12 (m, 1H, aromatic CH), 4.72 (s, 2H, ArCH₂ O), 4.48 (s, 1H, OH), 1.72 (m, 4H, pentyl CH₂), 0.90 (s, 9H, t-Bu), 0.64 (t, 6H, J=7.5, pentyl CH₃), 0.06 (s, 6H, SiMe₂).

EXAMPLE 157 3-(3-Pentyl)benzyl alcohol

A 250 mL round bottomed flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 3.9 g of 3-(3-hydroxy-3-pentyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol and 25 mL of CH₂ Cl₂. The solution was cooled to 0° C. in an ice bath and was treated with 15 mL of TFA followed by 5 mL of Et₃ SiH (Aldrich). After 30 min at 0° C. the ice bath was removed and the solution was stirred at RT for 18 h. The solution was concentrated to a viscous liquid. This material was dissolved in ether (60 mL), washed with 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous MgSO₄, and concentrated in vacuo. The resulting liquid was dissolved in 10 mL of THF and treated with 63 mL of 1M Bu₄ NF/THF (Aldrich). After stirring at RT for 18 h, the solution was concentrated and the resulting syrup mixed with 80 mL of water. The mixture was extracted with ether (4×30 mL). The combined extracts were washed with 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous MgSO₄, and concentrated to afford a light yellow liquid. Analysis by tlc (SiO₂, 9:1 hexane:EtOAc) indicated a major new component at R_(f) =0.30. The crude product was purified by flash chromatography on silica gel (85:15 hexane:EtOAc) to give 1.84 g (82%) of 3-(3-pentyl)benzyl alcohol as a clear liquid. ¹ H-NMR: (300 MHz, DMSO-d₆) δ 7.21 (t, 1H, J=7.5, aromatic CH), 7.10 (m, 2H, aromatic CH), 6.99 (d, 1H, J=7.4, aromatic CH), 5.12 (t, 1H, J=5.8, OH), 4.46 (d, 2H, J=5.8, ArCH₂ O), 2.26 (m, 1H, pentyl methine), 1.62 (m, 2H, pentyl CH₂), 1.49 (m, 2H, pentyl CH₂), 0.69 (t, 6H, J=7.4, pentyl CH₃).

EXAMPLE 158 3-(3-Pentyl)benzyl bromide

According to example 17, 1.84 g of 3-(3-pentyl)benzyl alcohol was treated with 0.34 mL of PBr₃ in 25 mL of anhydrous CH₂ Cl₂ to afford 2.23 g (90%) of 3-(3-Pentyl)benzyl bromide as a clear liquid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.26 (m, 3H, aromatic CH), 7.12 (m, 1H, aromatic CH), 4.70 (s, 2H, ArCH₂), 2.33 (m, 1H, pentyl methine), 1.78-1.40 (m, 4H, pentyl CH₂), 0.73 (t, 6H, J=7.3, pentyl CH₃).

EXAMPLE 159 tert-Butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)-4-morpholinecarboxylate

According to example 142, 3.27 g of tert-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate was alkylated with 2.23 g of 3-(3-pentyl)benzyl bromide in 70 mL of anhydrous THF using 9.71 mL of 1 M sodium bis(trimethylsilyl)amide in THF. The crude product was subjected to flash chromatography on silica gel (9:1 hexane:EtOAc) and then recrystallization from EtOH-water to afford 2.26 g (48%) of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl-4-morpholinecarboxylate as a white crystalline solid, m.p. 143-144° C.

EXAMPLE 160 (2R, 3S, 5S)-6-Oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)morpholine

To a 250 mL round bottomed flask equipped with a magnetic stirrer and a nitrogen inlet were added 2.19 g of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)-4-morpholinecarboxylate and 45 mL of anhydrous CH₂ Cl₂. The solution was cooled to 0° C. and was treated with 3.5 mL of TFA. The solution was allowed to warm to RT and was stirred under nitrogen. After 5 h, tlc (SiO₂, 9:1 hexane:EtOAc) indicated a trace of starting material and a major new component at R_(f) =0.42. The reaction mixture was poured into 150 mL of 6% aqueous NaHCO₃ and stirred for several minutes. The phases were separated and the aqueous portion was extracted with 3 additional 40 mL portions of CH₂ Cl₂. The extracts were combined with the original CH₂ Cl₂ solution and washed with 5% aqueous NaHCO₃ (3×60 mL. The solution was dried over anhydrous MgSO₄ and concentrated to give a light tan solid. This material was purified by flash chromatography on silica gel (85:15 hexane:EtOAc) to afford 1.47 g (84%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)morpholine as a white crystalline solid. An analytical sample was prepared by recrystallization from EtOH--H₂ O. Melting point, 96-97° C.

EXAMPLE 161 3-(3-Pentyl)-L-phenylalanine

To a 300 mL Parr bottle were added 0.41 g of PdCl₂, 1.36 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)morpholine, 50 mL of EtOH, and 30 mL of THF. The mixture was deoxygenated by bubbling nitrogen through for 10 min and was hydrogenated at 45 psi for 18 h. The vessel was purged with nitrogen, catalyst removed by filtration through celite, and the filtrate concentrated in vacuo to a volume of 15 mL. The solution was chilled in an ice bath and triturated with additional of 50 mL of hexane. A fine precipitate slowly formed. After storing at 5° C. overnight, the solid was collected by filtration and dried in vacuo to afford 0.65 g (72%) of 3-(3-pentyl)-L-phenylalanine.HCl as a white powder, m.p. 170° C. (dec).

EXAMPLE 162 tert-Butyl 3-(3-pentyl)-L-phenylalaninate

According to example 54, 0.60 g of 3-(3-pentyl)-L-phenylalanine.HCl was treated with 30 mL of isobutylene in 35 mL of 1,4-dioxane in the presence of 0.5 mL of conc. H₂ SO₄. Work-up in the usual manner followed by acidification with 2 mL of 1N ethereal HCl afforded 0.53 g (74%) of tert-butyl 3-(3-pentyl)-L-phenylalaninate.HCl as a light yellow glass.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 8.63 (br s, 3H, NH₃ ⁺), 7.28 (t, 1H, J=7.45, aromatic CH), 7.10 (m, 3H, aromatic CH), 4.12 (m, 1H, methine), 3.23 (dd; 1H; J=13.9, 5.1; ArCH₂), 2.96 (dd; 1H; J=13.9, 9.1; ArCH₂), 2.32 (m, 1H, pentyl methine), 1.78-1.40 (m, 4H, pentyl CH₂), 1.28 (s, 9H, t-Bu), 0.74 (t, 6H, J=7.3, pentyl CH₃).

EXAMPLE 163 tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate

According to example 145, 0.53 g of tert-butyl 3-(3-pentyl)-L-phenylalaninate.HCl was coupled with 0.55 g of N-Cbz-L-glutamic acid γ-tert-butyl ester using 0.33 g of EDC and 0.18 mL of N-methylmorpholine in 15 mL of anhydrous CH₂ Cl₂. The crude product was purified by flash chromatography on silica gel (8:2 hexane:EtOAc) to afford 0.80 g (81%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate as a white crystalline solid, m.p. 50-53° C.

EXAMPLE 164 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate

According to example 48, 0.72 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate was hydrogenolyzed using 0.12 g of 10% Pd(C) in 70 mL of MeOH. This afforded 0.51 g (91%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for ¹ H-NMR analysis was prepared by treating 5 mg of the product with excess 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.03 (d, 1H, J=6.9, NH), 8.38 (br s, 3H, NH₃ ⁺), 7.23 (t, 1H, J=7.2, aromatic CH), 7.06 (m, 3H, aromatic CH), 4.39 (m, 1H, methine), 3.87 (m, 1H, methine), 3.01 (d, 2H, J=7.4, ArCH₂), 2.35 (m, 3H, pentyl methine glu 4-CH₂), 2.01 (m, 2H, glu 3-CH₂), 1.74-1.48 (m, 4H, pentyl CH₂), 1.41 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu), 0.73 (t, 6H, J=7.3, pentyl CH₃).

EXAMPLE 165 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate

According to example 49, 0.199 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate was coupled with 0.25 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate using 0.24 mL of DECP and 0.26 mL of Et₃ N in 25 mL of DMF. The crude product was subjected to flash chromatography twice on silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH; 95:5CH₂ Cl₂ :MeOH) to afford 0.215 g (52%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate as a yellow powder, m.p. 175° C. (dec).

EXAMPLE 166 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-(3-pentyl)-L-phenylalanine

According to example 50, 0.184 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-(3-pentyl)-L-phenylalaninate was treated with gaseous HCl in 25 mL of CH₃ NO₂ for 45 min. The yellow suspension was concentrated in vacuo to dryness. Analysis of the product by HPLC (C18, 65:35:0.1 H₂ O:MeCN:TFA) indicated a major component at k'=1.61 and a minor component at k'=0.25. The crude product was purified by semi-preparative HPLC (C18, 65:35:0.1 H₂ O:MeCN:TFA). Fractions containing pure product (analytical HPLC) were combined and subjected to rotary evaporation to remove CH₃ CN. A yellow suspension resulted which was frozen and lyophilized to afford 0.12 g (58%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-(3-pentyl)-L-phenylalanine as a yellow powder, m.p. 120° C. (dec).

HPLC: one peak on C18, k'=1.61, 65:35:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.87-11.90 (br m, 2H, COOH), 9.20 (br s, 1H, NH₂), 9.00 (br s, 1H, NH₂), 8.70 (s, 1H, pteridinyl 7-CH), 8.60-8.23 (br m, 2H, NH₂), 8.11 (d, 1H, J=7.6, amide NH), 7.96 (d, 1H, J=7.8, amide NH), 7.70 (d, 2H, J=8.7, aromatic CH), 7.07 (m, 1H, aromatic CH), 6.98 (d, 2H, J=7.9, aromatic CH), 6.90 (d, 1H, J=7.6, aromatic CH), 6.80 (d, 2H, J=8.6, aromatic CH), 4.86 (s, 2H, pteridinyl-CH₂), 4.41 (m, 2H, methines), 3.23 (s, 3H, N--CH₃), 2.92 (m, 2H, ArCH₂), 2.21 (m, 3H, pentyl methine, glu 4-CH₂), 2.03-1.77 (m, 2H, glu 3-CH₂), 1.68-1.37 (m, 4H, pentyl CH₂), 0.62 (m, 6H, pentyl CH₃).

Elemental Analysis: Calcd. for C₃₄ H₄₁ N₉ O₆.1.5TFA.1.8H₂ O (MW 875.22): C, 50.78; H, 5.31; N, 14.40. Found: C, 50.76; H, 5.40; N, 14.34.

Mass Spectrum: (Ion Spray) 672 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 259.8 (20000), 308.7 (19800), 373.7 (5890). λ_(min) (ε) 241.5 (11600), 275.0 (13200), 345.8 (4420).

EXAMPLE 167 2-Iodo-4-nitro-L-phenylalanine

A 250 mL round bottomed flask equipped with a magnetic stirrer was charged with 60 mL of fuming sulfuric acid (Aldrich) and 12.1 g of iodine (Aldrich). To the resulting dark red solution was added 10.0 g of 4-nitro-L-phenylalanine by slow addition. The flask was fitted with a CaCl₂ drying tube and the reaction mixture was stirred at RT for 18 h. The mixture was then poured onto 300 g of crushed ice. A yellow-brown emulsion resulted which was treated with 7.4 g of sodium thiosulfate and stirred. The resulting light brown suspension was basified to pH=9 by addition of solid NaCO₃. This was followed by acidification to pH=5.5 by addition of conc. H₃ SO₄. A brown solid precipitated which was collected by filtration and dried in vacuo. This afforded 6.54 g (41%) of 2-iodo-4-nitro-L-phenylalanine as a brown powder, m.p.>200° C.

¹ H-NMR: (200 MHz, DMSO-d₆ /TFA) d 8.62 (s, 1H, aromatic CH), 8.46 (br s, 3H, NH₃ ⁺), 8.26 (d, 1H, J=8.5, aromatic CH), 7.65 (d, 1H, J=8.5, aromatic CH), 4.22 (m, 1H, methine), 3.34 (m, 2H, ArCH₂).

EXAMPLE 168 Ethyl 2-iodo-4-nitro-L-phenylalaninate

According to example 8, 1.40 g of 2-iodo-4-nitro-L-phenylalanine was esterified by refluxing in 50 mL of ethanolic HCl for 18 h. Work-up in the usual manner followed by flash chromatography on silica gel (95:5 CH₂ Cl₂ :MeOH) afforded 1.19 g (78%) of ethyl 2-iodo-4-nitro-L-phenylalaninate as a yellow crystalline solid, m.p. 33-34° C.

EXAMPLE 169 Ethyl N-((benzyloxy)carbonyl)-2-iodo-4-nitro-L-phenylalaninate

To a 250 mL round bottomed flask equipped with a magnetic stirrer and a nitrogen inlet were added 4.7 g of ethyl 2-iodo-4-nitro-L-phenylalaninate, 125 mL of 1,4-dioxane, and 2.2 mL of Et₃ N. The solution was cooled in an ice water bath and treated with 2.2 mL of benzyl chloroformate by slow addition. The ice bath was removed and the reaction mixture was stirred at RT for 18 h. The mixture was filtered and the filtrate was concentrated to dryness. The brown residue was dissolved in EtOAc, washed with 5% aqueous NaHCO₃ (4×75 mL), dried over anhydrous MgSO₄ and concentrated in vacuo. Analysis of the crude product by tlc (SiO₂, 75:25 hexane:EtOAc) indicated a major new component at R_(f) =0.35 and minor components at R_(f) =0.40 and 0.25. The R_(f) =0.35 product was isolated by flash chromatography on silica gel (75:25 hexane:EtOAc). This afforded 2.08 g (32%) of ethyl N-((benzyloxy)carbonyl)-2-iodo-4-nitro-L-phenylalaninate as a white powder, m.p. 85-87° C.

EXAMPLE 170 Ethyl 4-amino-2-cyclopentyl-L-phenylalaninate

According to example 151, 1.65 g of ethyl N-((benzyloxy)carbonyl)-2-iodo-4-nitro-L-phenylalaninate was subjected to Heck coupling with 20 mL of cyclopentene using 60 mL of toluene, 0.074 g of Pd(OAc)₂, 0.20 g of tri-ortho-tolylphosphine, and 0.51 mL of Et₃ N. The reaction was carried out at 110° C. for 24 h. Analysis of the crude product by tlc (SiO₂, 8:2 hexane:EtOAc) indicated a major product at R_(f) =0.40 and minor components at R_(f) =0.38, 0.60, and 0.95. The R_(f) =0.40 material was isolated by flash chromatography on silica gel (8:2 hexane:EtOAc). This afforded 1.03 g (71%) of the desired olefin mixture as a clear liquid. This material was subjected to catalytic hydrogenation at 45 psi for 18 h using 55 mL of MeOH and 0.25 g of 10% Pd(C). The crude product was purified by flash chromatography (SiO₂, 99:1 EtOAc:MeOH) to give 0.56 g (61% for both steps) of ethyl 4-amino-2-cyclopentyl-L-phenylalaninate as a thick transparent oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 6.72 (d, 1H, J=8.2, aromatic CH), 6.50 (d, 1H, J=2.3, aromatic CH), 6.29 (dd; 1H; J=8.1, 2.3; aromatic CH), 4.80 (br s, 2H, aromatic NH₂), 4.00 (q, 2H, J=7.1, ethyl CH₂), 3.12 (m, 1H, cyclopentyl methine), 2.72 (m, 2H, ArCH₂), 2.03-1.37 (m, 10H, cyclopentyl CH₂, NH₂), 1.11 (t, 3H, J=7.1, ethyl CH₃).

EXAMPLE 171 Ethyl 2-cyclopentyl-L-tyrosinate

To a 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer were added a solution of 0.56 g of ethyl 4-amino-2-cyclopentyl-L-phenylalaninate in 60 mL of 0.1 M aqueous H₂ SO₄. After cooling to 3° C., the solution was treated with a solution of 0.148 g of sodium nitrite (Aldrich) in 10 mL of water. The solution was stirred at 3° C. for 10 min and was then treated with 95 mL of 1.5 M aqueous CuSO₄ followed by 0.25 g of Cu₂ O (Aldrich). The mixture was heated to 45° C. for 35 min with stirring. Gas evolution was evident during this period. The mixture was cooled to RT and filtered. The blue filtrate was treated with 1N aqueous NaOH to pH=5.5. The resulting blue-green suspension was transfered to a separatory funnel and extracted with CHCl₃ (5×100 mL). The combined CHCl₃ extracts were dried over MgSO₄ and concentrated to give 0.12 g of a green oil. The blue-green suspension was then treated with solid NaHCO₃ until gas evolution ceased and was extracted with CHCl₃ a second time (4×50 mL). The combined extracts were washed with water, dried (MgSO₄), and concentrated to give 0.076 g of an oil. This material was combined with the original 0.12 g of product and purified by flash chromatography (SiO₂, 95:5 CH₂ Cl₂ :MeOH) to afford 0.168 g (30%) of ethyl 2-cyclopentyl-L-tyrosinate as a thick, transparent oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.07 (s, 1H, OH), 6.87 (d, 1H, J=8.2, aromatic CH), 6.66 (d, 1H, J=2.4, aromatic CH), 6.48 (dd; 1H; J=8.1, 2.4; aromatic CH), 4.00 (q, 2H, J=7.1, ethyl CH₂), 3.16 (m, 1H, cyclopentyl methine), 2.78 (m, 2H, ArCH₂), 2.05-1.32 (m, 10H, cyclopentyl CH₂, NH₂), 1.10 (t, 3H, J=7.1, ethyl CH₃).

EXAMPLE 172 Ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate

According to example 145, 0.153 g of ethyl 2-cyclopentyl-L-tyrosinate was coupled with 0.171 g of N-Cbz-L-glutamic acid γ-ethyl ester using 0.111 g of EDC in 8 mL of CH₂ Cl₂ for 4.5 h. The crude product was purified by flash chromatography twice on silica gel (1:1 hexane:EtOAc; 95:5 CH₂ Cl₂ :MeOH) to afford 0.245 g (78%) of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate as a colorless foam.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 9.09 (s, 1H, OH), 8.37 (d, 1H, J=7.3 NH), 7.33 (m, 6H, aromatic CH, NH), 6.88 (d, 1H, J=8.4, aromatic CH), 6.63 (s, 1H, aromatic CH), 6.43 (d, 1H, J=8.3, aromatic CH), 4.99 (m, 2H, PhCH₂ O), 4.28 (m, 1H, methine), 4.00 (m, 5H, ethyl CH₂, methine), 3.09 (m, 1H, cyclopentyl methine), 2.96 (m, 1H, ArCH₂), 2.83 (m, 1H, ArCH₂), 2.29 (t, 2H, J=7.9, glu 4-CH₂), 2.02-1.53 (m, 8H, glu 3-CH₂, cyclopentyl CH₂), 1.46 (m, 2H, cyclopentyl CH₂), 1.15 (t, 3H, J=7.1, ethyl CH₃), 1.04 (t, 3H, J=7.1, ethyl CH₃).

EXAMPLE 173 Ethyl 5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate

According to example 48, 0.24 g of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate was hydrogenolyzed using 0.042 g of 10% Pd(C) in 85 mL of MeOH. The crude product was purified by flash chromatography (SiO₂, 95:5 CH₂ Cl₂ :MeOH) to afford 0.154 g (84%) of ethyl 5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate as a thick transparent oil.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 9.08 (s, 1H, OH), 8.23 (d, 1H, J=7.9, NH), 6.87 (d, 1H, J=8.1, aromatic CH), 6.63 (d, 1H, J=2.1, aromatic CH), 6.44 (dd; 1H; J=8.1, 2.1; aromatic CH), 4.31 (m, 1H, methine), 4.00 (m, 4H, ethyl CH₂), 3.12 (m, 2H, glu methine cyclopentyl methine), 2.98 (m, 1H, ArCH₂), 2.82 (m, 1H, ArCH₂), 2.27 (t, 2H, J=7.9, glu 4-CH₂), 2.00-1.35 (m, 12H, glu 3-CH₂, NH₂, cyclopentyl CH₂), 1.07 (t, 3H, J=7.1, ethyl CH₃), 1.16 (t, 3H, J=7.1, ethyl CH₃).

EXAMPLE 174 Ethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate

According to example 49, 0.131 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate was coupled with 0.15 g of ethyl 5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate using 0.08 mL of DECP and 0.10 mL of Et₃ N in 12 mL of DMF. The crude product was subjected to flash chromatography twice on silica gel (93:7 CH₂ Cl₂ :MeOH; 92:8 CH₂ Cl₂ :MeOH) to afford 0.134 g (52%) of ethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate as a yellow powder, m.p. 135-150° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 9.06 (s, 1H, OH), 8.54 (s, 1H, pteridinyl 7-CH), 8.34 (d, 1H, J=7.4, amide NH), 7.96 (d, 1H, J=8.2, amide NH), 7.70 (d, 2H, J=8.6, aromatic CH), 7.63 (br s, 1H, NH₂), 7.43 (br s, 1H, NH₂), 6.86 (d, 1H, J=8.4, aromatic CH), 6.80 (d, 2H, J=8.9, aromatic CH), 6.60 (m, 3H, aromatic CH, NH₂), 6.40 (dd; 1H; J=8.2, 1.9; aromatic CH), 4.77 (s, 2H, pteridinyl-CH₂), 4.42 (m, 1H, methine), 4.25 (m, 1H, methine), 4.07-3.89 (m, 4H, ethyl CH₂), 3.19 (s, 3H, N--CH₃), 3.08 (m, 1H, cyclopentyl methine), 2.94 (m, 1H, ArCH₂), 2.82 (m, 1H, ArCH₂), 2.31 (t, 2H, J=7.9, glu 4-CH₂), 2.04-1.32 (m, 10H, glu 3-CH₂, cyclopentyl CH₂), 1.13 (t, 3H, J=7.1, ethyl CH₃), 1.02 (t, 3H, J=7.1, ethyl CH₃).

EXAMPLE 175 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-tyrosine

A solution of 0.130 g of ethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-1-yl-2-cyclopentyl-L-tyrosinate in 10 mL of 1:1 THF:H₂ O was treated with 17 mg of LiOH and the solution was stirred at RT under nitrogen. The hydrolysis was monitored by HPLC (C18, 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min). An additional 10 mg portion of LiOH was added after 3 h. Analysis by HPLC (above conditions) 2 h after the second LiOH addition indicated a single component at k'=0.62. The solution was neutralized by addition of 1N HCl. A yellow precipitate resulted which was collected by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, frozen, and lyophilized to afford 0.106 g (83%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-tyrosine as a yellow powder, m.p. 185° C. (dec).

HPLC: one peak on C18, k'=2.10, 75:25:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.75-11.85 (br m, COOH), 9.03 (br s, 1H, OH), 8.56 (s, 1H, pteridinyl 7-CH), 8.13 (d, 1H, J=7.9, amide NH), 7.96 (d, 1H, J=7.8, amide NH), 7.72 (br s, 1H, NH₂), 7.70 (d, 2H, J=8.7, aromatic CH), 7.53 (br s, 1H, NH₂), 6.89 (d, 1H, J=8.4, aromatic CH), 6.80 (m, 3H, aromatic CH, NH₂), 6.69 (br s, 1H, NH₂), 6.60 (s, 1H, aromatic CH), 6.38 (dd; 1H; J=8.3, 2.2; aromatic CH), 4.78 (s, 2H, pteridinyl-CH₂), 4.41 (m, 1H, methine), 4.22 (m, 1H, methine), 3.21 (s, 3H, N--CH₃), 3.12 (m, 1H, cyclopentyl methine), 3.00 (dd; 1H; J=14.3, 5.3; ArCH₂), 2.76 (dd; 1H; J=14.3, 8.8; ArCH₂), 2.23 (t, 2H, J=7.9, glu 4-CH₂), 2.05-1.30 (m, 10H, glu 3-CH₂, cyclopentyl CH₂).

Elemental Analysis: Calcd. for C₃₄ H₃₉ N₉ O₇.2.5H₂ O (MW 730.78): C, 55.88; H, 6.07; N, 17.25. Found: C, 55.91; H, 5.94; N, 17.10.

Mass Spectrum: (Ion Spray) 686 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 259.0 (25000), 308.2 (25500), 371.5 (7960). λ_(min) (ε) 242.3 (16000), 273.0 (17700), 346.8 (6350). sh (e) 220.9 (30100), 288.1 (21000).

EXAMPLE 176 tert-Butyl 3,5-diiodo-L-tyrosinate

A solution of 5.0 g of 3,5-diiodo-L-tyrosine and 0.95 mL of 70% aqueous HClO₄ in 155 mL of tert-butyl acetate was stirred at RT under nitrogen for 4 days. The solution was neutralized by addition of 150 mL of 5% aqueous NaHCO₃ and the mixture was subjected to rotary evaporation to remove tert-butyl acetate. An off-white precipitate resulted which was collected by filtration and dried in vacuo. The crude product was subjected to flash chromatography on silica gel (97:3 CH₂ Cl₂ :MeOH) to afford 4.2 g (75%) of tert-butyl 3,5-diiodo-L-tyrosinate as a white powder, m.p. 165-168° C.

EXAMPLE 177 tert-Butyl N-(9-fluorenylmethyloxycarbonyl)-5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate

According to example 145, 1.50 g of tert-butyl 3,5-diiodo-L-tyrosinate was coupled with 1.31 g of N-FMOC-L-glutamic acid γ-tert-butyl ester using 0.62 g of EDC in 45 mL of CH₂ Cl₂ for 3 h. The crude product was purified by flash chromatography on silica gel (7:3 hexane:EtOAc) to afford 1.78 g (65%) of tert-butyl N-(9-fluorenylmethyloxycarbonyl)-5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate as a light brown foam.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.41 (s, 1H, OH), 8.27 (d, 1H, J=7.0, NH), 7.91 (d, 2H, J=7.2, aromatic CH), 7.75 (m, 2H, aromatic CH), 7.61 (s, 2H, aromatic CH), 7.57-7.28 (m, 5H, aromatic CH, NH), 4.26 (m, 4H, fluorenyl-CH₂ O, fluorenyl methine, α-methine), 4.08 (m, 1H, α-methine), 2.83 (d, 2H, J=7.4, ArCH₂), 2.25 (t, 2H, J=7.9, glu 4-CH₂), 1.83 (br m, 2H, glu 3-CH₂), 1.41 (s, 9H, t-Bu), 1.33 (s, 9H, t-Bu).

EXAMPLE 178 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate

To a solution of 1.66 g of tert-butyl N-(9-fluorenylmethyloxycarbonyl)-5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate in 7 mL of anhydrous DMF were added 1.71 mL of piperidine (Sigma). After stirring at RT under nitrogen for 1 h, the solution was mixed with 75 mL of water and was extracted with CH₂ Cl₂. The combined CH₂ Cl₂ solutions were washed with aqueous NaCl (4×30 mL), dried over MgSO₄, and concentrated in vacuo to give a white solid. Analysis of the crude product by tlc (SiO₂, 95:5 CH₂ Cl₂ :MeOH) indicated major new components at R_(f) =0.4 and 0.5. The R_(f) =0.4 material was isolated by flash chromatography on silica gel (95:5 CH₂ Cl₂ :MeOH) to afford 1.1 g (87%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate as a white foam, m.p. 45-57° C. A sample of the corresponding HCl salt was prepared for NMR by treating 10 mg of product with 1N ethereal HCl and concentrating to dryness.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.42 (br s, 1H, OH), 8.98 (d, 1H, J=7.0, NH), 8.33 (br s, 3H, NH₃ ⁺), 7.67 (s, 2H, aromatic CH), 4.36 (m,1H, methine), 3.88 (m, 1H, methine), 2.86 (d, 2H, J=7.0, ArCH₂), 2.38 (m, 2H, glu 4-CH₂), 2.01 (m, 2H, glu 3-CH₂), 1.41 (s, 9H, t-Bu), 1.35 (s, 9H, t-Bu).

EXAMPLE 179 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate

According to example 154, 0.25 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate was coupled with 0.132 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate using 0.06 mL of DECP, 0.07 mL of Et₃ N, and 10 mL of DMF. The crude product was purified by flash chromatography on silica gel (95:5 CH₂ Cl₂ :MeOH) to afford 0.18 g (50%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate as a yellow powder, m.p. 172° C. (dec).

EXAMPLE 180 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3,5-diiodo-L-tyrosine

According to example 50, 0.119 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3,5-diiodo-L-tyrosinate was treated with gaseous HCl in 15 mL of CH₃ NO₂ for 30 min. The yellow suspension was concentrated in vacuo to dryness. Analysis of the product by ¹ H-NMR and reverse phase HPLC indicated the desired product contaminated with minor impurities. The mixture was purified by semi-preparative HPLC (C18, 77:23:0.1 H₂ O:MeCN:TFA). Fractions containing pure product were combined and subjected to rotary evaporation to remove MeCN. The resulting suspension was frozen and lyophilized to afford 0.024 g (18%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3,5-diiodo-L-tyrosine as a yellow powder, m.p. 205° C. (dec).

HPLC: one peak on C18, k'=2.74, 77:23:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 13.20-11.90 (br m, COOH), 9.35 (s, 1H, OH), 9.21 (br s, 1H, NH₂), 9.02 (br s, 1H, NH₂), 8.70 (s, 1H, pteridinyl 7-CH), 8.52 (br s, 1H, NH₂), 8.13 (d, 1H, J=7.6, amide NH), 7.98 (d, 1H, J=7.9, amide NH), 7.72 (d, 2H, J=9.0, aromatic CH), 7.57 (s, 2H, aromatic CH), 7.51 (br s, 1H, NH₂), 6.80 (d, 2H, J=8.9, aromatic CH), 4.86 (s, 2H, pteridinyl-CH₂), 4.41 (m, 1H, methine), 4.30 (m, 1H, methine), 3.23 (s, 3H, N--CH₃), 2.81 (m, 2H, ArCH₂), 2.25 (t, 2H, J=7.6, glu 4-CH₂), 1.90 (m, 2H, glu 3-CH₂).

Elemental Analysis: Calcd. for C₂₉ H₂₉ N₉ O₇ I₂.1.7 TFA.2.3 H₂ O (MW 1104.69): C, 35.23; H, 3.22; N, 11.41. Found: C, 35.25; H, 3.24; N, 11.44.

Mass Spectrum: (Ion Spray) 870 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 219.7 (48900), 258.0 (26500), 306.0 (26200), 373.3 (7780). λ_(min) (ε) 214.9 (48300), 246.0 (23700), 273.7 (18400), 347.6 (6200).

EXAMPLE 181 Methyl 4-hydroxy-3-iodobenzoate

To a 500 mL 3-necked flask equipped with a magnetic stirrer, a thermometer, and a condenser were added 10.0 g of methyl 4-hydroxybenzoate (Aldrich) and 30 mL of glacial acetic acid. To this suspension was added a solution of 11.74 g of iodine monochloride in 30 mL of glacial acetic acid via addition funnel. The solution was heated at 80° C. for 1 h, 45° C. for 18 h, and 90° C. for an additional 4 h. Cooling to RT afforded a thick red-orange suspension which was transferred to a 1 L separatory funnel and dissolved in 500 mL of CHCl₃ by shaking vigorously. The dark purple solution was washed with water (2×100 mL), saturated aqueous sodium thiosulfate (2×100 mL), and 5% aqueous NaHCO₃ (2×100 mL). A light yellow solution resulted which was dried over MgSO₄, and concentrated in vacuo to give a white solid. This material was recystallized from water-MeOH to afford 14.92 g (82%) of methyl 4-hydroxy-3-iodobenzoate as a white crystalline solid, m.p. 145-146° C.

EXAMPLE 182 Methyl 4-hydroxy-3-cyclopentylbenzoate

According to example 151, 5.0 g of methyl 4-hydroxy-3-iodobenzoate was subjected to Heck coupling with 25 mL of cyclopentene using 80 mL of toluene, 0.40 g of Pd(OAc)₂, 1.10 g of tri-ortho-tolylphosphine, and 2.76 mL of Et₃ N. The reaction was carried out at 110° C. for 24 h. Analysis of the crude product by tlc (SiO₂, 75:25 hexane: EtOAc) indicated a major new component at R_(f) =0.50. This material was isolated by flash chromatography on silica gel (75:25 hexane:EtOAc). This gave 2.97 g (76%) of the desired olefin mixture (¹ H-NMR) as a brown solid. This material was subjected to catalytic hydrogenation at 45 psi for 18 h using 80 mL of MeOH and 1.0 g of 10% Pd(C). This afforded 2.59 g (65% for both steps) of methyl 4-hydroxy-3-cyclopentylbenzoate as a white solid, m.p. 114-116° C.

EXAMPLE 183 Methyl 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzoate

A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 1.79 g of methyl 4-hydroxy-3-cyclopentylbenzoate, 1.22 g of imidazole (Aldrich), 30 mL of anhydrous DMF, and 1.47 g of tert-butyldimethylsilyl chloride (Aldrich). The solution was stirred at RT under nitrogen for 24 h and then concentrated in vacuo to give a thick oil. This material was subjected to flash chromatography on silica gel (95:5 hexane:EtOAc) to afford 2.43 g (89%) of methyl 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzoate as a clear liquid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.81 (d, 1H, J=2.0, aromatic CH), 7.72 (dd; 1H; J=8.4, 2.0; aromatic CH), 6.93 (d, 1H, J=8.4, aromatic CH), 3.80 (s, 3H, CH₃), 3.30 (m, 1H, cyclopentyl methine), 2.06-1.37 (m, 8H, cyclopentyl CH₂), 0.98 (s, 9H, t-Bu), 0.27 (s, 6H, SiMe₂).

EXAMPLE 184 4-((tert-Butyldimethylsilyl)oxy)-3-cyclopentylbenzyl alcohol

To a 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 2.58 g of methyl 4-((tert-butyldimethylsilyl)oxy-3-cyclopentylbenzoate and 15 mL of anhydrous toluene. The solution was cooled in an ice water bath and was treated with 19.3 mL of 1M diisobutylaluminum hydride in toluene (Aldrich). The solution was stirred at 0° C. for 30 min and was allowed to warm to RT. After 2 h at RT the solution was poured into 130 mL of saturated aqueous potassium sodium tartrate and the resulting mixture was stirred for 20 min. The mixture was then extracted with ethyl ether (3×70 mL). The combined ether extracts were washed with water (3×70 mL), dried over anhydrous MgSO₄, and concentrated to give a cloudy oil. The crude product was purified by flash chromatography on silica gel (8:2 hexane:EtOAc) to afford 1.94 g (82%) of 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzyl alcohol as a clear liquid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.18 (d, 1H, J=2.0, aromatic CH), 7.02 (dd; 1H; J=8.2, 2.2; aromatic CH), 6.76 (d, 1H, J=8.0, aromatic CH), 5.02 (t, 1H, J=5.8, OH), 4.40 (d, 2H, J=5.6, ArCH₂ O), 3.29 (m, 1H, cyclopentyl methine), 2.04-1.38 (m, 8H, cyclopentyl CH₂), 1.03 (s, 9H, t-Bu), 0.22 (s, 6H, SiMe₂).

EXAMPLE 185 4-((tert-Butyldimethylsilyl)oxy)-3-cyclopentylbenzaldehyde

A solution of 1.94 g of 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzyl alcohol in 70 mL of anhydrous CH₂ Cl₂ was treated with 2.05 g of pyridinium chlorochromate (Aldrich) and was stirred at RT under nitrogen. After 30 min, tlc (SiO₂, 85:15 hexane:EtOAc) indicated no remaining starting material and a major new component at R_(f) =0.80. The mixture was filtered to remove solids and the filtrate was washed with 5% aqueous citric acid (4×50 mL) followed by 5% aqueous NaHCO₃ (4×50 mL). The CH₂ Cl₂ solution was dried over anhydrous MgSO₄ and concentrated in vacuo to give a brown oil. This material was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 1.52 g (79%) of 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzaldehyde as a clear oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.88 (s, 1H, aldehyde CH), 7.80 (d, 1H, J=2.0, aromatic CH), 7.69 (dd; 1H; J=8.2, 2.0; aromatic CH), 7.03 (d, 1H, J=8.3, aromatic CH), 3.29 (m, 1H, cyclopentyl methine), 2.08-1.43 (m, 8H, cyclopentyl CH₂), 1.01 (s, 9H, t-Bu), 0.30 (s, 6H, SiMe₂).

EXAMPLE 186 tert-Butyl trans-4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylcinnamate

To a 100 mL 3-necked flask equipped with a magnetic stirrer, a nitrogen inlet and a reflux condenser were added 1.57 g of tert-butyl diethylphosphonoacetate (Aldrich) and 20 mL of anhydrous ethyl ether. This was followed by addition of 0.40 g of 60% NaH mineral oil dispersion. The resulting cloudy solution was heated at reflux for 25 min, cooled to 0° C. and treated with a solution of 1.52 g of 4-((tert-butyldimethylsilyl)oxy-3-cyclopentylbenzaldehyde in 10 mL of ether. A thick yellow gel resulted which was broken up with a spatula. The mixture was allowed to warm to RT and stirring was continued for an additional 1 h. Analysis by tlc (SiO₂, 95:5 hexane:EtOAc) indicated a major new component at R_(f) =0.48 and minor components at R_(f) =0.06, 0.41 and 0.98. The mixture was diluted with 70 mL of ether, washed with saturated brine, and dried over anhydrous MgSO₄. The drying agent was removed by filtration and the filtrate concentrated to give a yellow oil. This material was purified by flash chromatography on silica gel (97:3 hexane:EtOAc) to afford 1.53 g (77%) of tert-butyl trans-4-((tert-butyldimethylsilyl)oxy-3-cyclopentylcinnamate as a clear oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.59-7.36 (m, 3H, aromatic CH, olefinic CH), 6.83 (d, 1H, J=8.4, aromatic CH), 6.38 (d, 1H, J=15.8, olefinic CH), 3.27 (m, 1H, cyclopentyl methine), 2.02-1.52 (m, 8H, cyclopentyl CH₂), 1.49 (s, 9H, t-Bu), 1.00 (s, 9H, Si-t-Bu), 0.25 (s, 6H, SiMe₂).

EXAMPLE 187 (2R, 3S)-tert-Butyl 3-(4-((tert-butyldimethylsilyl)oxy-3-cyclopentylphenyl)-2,3-dihydroxypropionate

To a 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 20 mL of tert-butyl alcohol, 20 mL of water, 5.32 g of AD-mix-α (Sharpless, K. B.; et al. J. Org. Chem., 1992, 57, 2768-2771; reagent purchased from Aldrich) and 0.36 g of methanesulfonamide (Aldrich). This was followed by addition of 1.53 g of tert-butyl trans-4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylcinnamate. The reaction mixture was stirred at RT under nitrogen. After 18 h, 5.7 g of sodium sulfite were added and the mixture was stirred for 30 min. The mixture was concentrated in vacuo to 15 mL, diluted with 100 mL of water and extracted with EtOAc (4×50 mL). The combined EtOAc extracts were washed with water (3×50 mL), dried over anhydrous MgSO₄ and concentrated to give a cloudy oil. This material was purified by flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 1.44 g (87%) of tert-butyl (2R, 3S)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)-2,3-dihydroxypropionate as a thick clear oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.16 (s, 1H, aromatic CH), 7.01 (d, 1H, J=8.2, aromatic CH), 6.73 (d, 1H, J=8.2, aromatic CH), 5.28 (d, 1H, J=5.4, OH), 5.25 (d, 1H, J=7.1, OH), 4.55 (t, 1H, J=5.6, propionate 2-methine), 3.93 (t, 1H, J=6.8, propionate 3-methine), 3.27 (m, 1H, cyclopentyl methine), 2.04-1.37 (m, 8H, cyclopentyl CH₂), 1.24 (s, 9H, t-Bu), 0.99 (s, 9H, Si-t-Bu), 0.21 (s, 6H, SiMe₂).

EXAMPLE 188 (R)-tert-Butyl 3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)-2-hydroxypropionate

To a 300 mL Parr bottle were added 0.35 g of 10% Pd(C) and 20 mL of tert-butyl alcohol under a nitrogen flush. To this was added a solution of 1.42 g of tert-butyl (2R, 3S)-3-(4-((tert-butyldimethylsilyl)oxy-3-cyclopentylphenyl)-2,3-dihydroxypropionate in 40 mL of tert-butyl alcohol. The mixture was deoxygenated by bubbling nitrogen through for 5 min while gently warming the bottle to avoid freezing of tert-butyl alcohol. The mixture was treated with 4 drops of concentrated HClO₄ and hydrogenated at 45 psi and 40° C. for 72 h. The vessel was purged with nitrogen, catalyst removed by filtration through celite, and the filtrate concentrated in vacuo to 20 mL. The solution was mixed with 60 mL of 5% aqueous NaHCO₃ and the mixture extracted with EtOAc (4×40 mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous MgSO₄ and concentrated to give a cloudy oil. This material was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 1.17 g (86%) of tert-butyl (R)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)-2-hydroxypropionate as a thick transparent oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.05 (d, 1H, J=2.1, aromatic CH), 6.92 (dd; 1H; J=8.4, 2.1; aromatic CH), 6.69 (d, 1H, J=8.2, aromatic CH), 5.33 (d, 1H, J=5.7, OH), 4.08 (m, 1H, methine), 3.23 (m, 1H, cyclopentyl methine), 2.78 (d, 2H, J=6.1, ArCH₂), 2.00-1.38 (m, 8H, cyclopentyl CH₂), 1.32 (s, 9H, t-Bu), 0.99 (s, 9H, Si-t-Bu), 0.20 (s, 6H, SiMe₂).

EXAMPLE 189 (S)-tert-Butyl 2-((N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)propionate

To a 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 1.14 g of tert-butyl (R)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)-2-hydroxypropionate, 20 mL of anhydrous THF, 1.01 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma), and 0.78 g of triphenylphosphine (Aldrich). The solution was cooled to 0° C. and was treated with 0.47 mL of diethyl azodicarboxylate (Aldrich) by dropwise addition. The solution was allowed to warm to RT and stirring under nitrogen was continued. After 3.5 h, tlc (SiO₂, 85:15 hexane:EtOAc) indicated a major new component at R_(f) =0.46 and some remaining alcohol starting material at R_(f) =0.51. The solution was again cooled to 0° C. and treated with an additional 0.18 g of N-Cbz-L-glutamic acid γ-tert-butyl ester, 0.14 g of triphenylphosphine, and 0.085 mL of diethyl azodicarboxylate. After warming to RT and stirring for 18 h tlc indicated only a trace of starting material. The solution was concentrated in vacuo to dryness and the crude product purified by flash chromatography (SiO₂, 85:15 hexane:EtOAc). This afforded 1.86 g (84%) of (S)-tert-butyl 2-((N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)propionate as a thick transparent oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.76 (d, 1H, J=8.0, NH), 7.34 (m, 5H, aromatic CH), 7.11 (s, 1H, aromatic CH), 6.96 (d, 1H, J=8.1, aromatic CH), 6.68 (d, 1H, J=8.1, aromatic CH), 5.01 (m, 3H, PhCH₂ O, propionate methine), 4.19 (m, 1H, glu methine), 3.21 (m, 1H, cyclopentyl methine), 3.01 (d, 2H, J=5.1, ArCH₂), 2.32 (t, 2H, J=7.8, glu 4-CH₂), 2.12-1.43 (m, 10H, glu 3-CH₂, cyclopentyl CH₂), 1.39 (s, 9H, t-Bu), 1.26 (s, 9H, t-Bu), 0.98 (s, 9H, Si-t-Bu), 0.19 (s, 9H, SiMe₂).

EXAMPLE 190 (S)-tert-Butyl 2-((N-(benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate

A solution of 1.85 g of (S)-tert-butyl 2-((N-(benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(4-((tert-butyldimethylsilyl)oxy-3-cyclopentylphenyl)propionate in 20 mL of anhydrous THF was treated with 2.63 mL of 1M tetrabutylammonium fluoride/THF (Aldrich) and was stirred at 0° C. under nitrogen. After 15 min, tlc (SiO₂, 75:25 hexane:EtOAc) indicated no remaining starting material and a single new component at R_(f) =0.41. The solution was mixed with 80 mL of water and was extracted with EtOAc (4×40 mL). The combined EtOAc extracts were washed with saturated brine (3×60 mL), dried over anhydrous MgSO₄ and concentrated in vacuo to give a clear oil. This material was subjected to flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 1.5 g (96%) of (S)-tert-butyl 2-((N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate as a white foam.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.11 (s, 1H, OH), 7.77 (d, 1H, J=8.2, NH), 7.33 (m, 5H, aromatic CH), 7.01 (d, 1H, J=1.6, aromatic CH), 8.85 (dd; 1H; J=8.2, 1.7; aromatic CH), 6.68 (d, 1H, J=8.2, aromatic CH), 5.04 (s, 2H, PhCH₂ O), 4.97 (m, 1H, propionate methine), 4.18 (m, 1H, glu methine), 3.17 (m, 1H, cyclopentyl methine), 2.96 (d, 2H, J=6.0, ArCH₂), 2.34 (t, 2H, J=7.4, glu 4-CH₂), 2.13-1.43 (m, 10H, glu 3-CH₂, cyclopentyl CH₂), 1.40 (s, 9H, t-Bu), 1.29 (s, 9H, t-Bu).

EXAMPLE 191 (S)-tert-Butyl 2-((5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate

According to example 48, 1.49 g of (S)-tert-butyl 2-((N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate was hydrogenolyzed using 0.24 g of 10% Pd(C) in 70 mL of MeOH. Analysis of the crude product by tlc (SiO₂, 4:6 hexane:EtOAc) indicated a major component at R_(f) =0.47 and minor components at R_(f) =0.60 and 0.15. The crude product was purified by flash chromatography on silica gel (4:6 hexane:EtOAc) to afford 0.76 g (65%) of (S)-tert-butyl 2-((5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate as a white foam.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.12 (s, 1H, OH), 7.00 (s, 1H, aromatic CH), 6.86 (d, 1H, J=8.2, aromatic CH), 6.68 (d, 1H, J=8.2, aromatic CH), 4.92 (t, 1H, J=6.2, propionate methine), 4.04 (q, 1H, J=7.0, glu methine), 3.19 (m, 1H, cyclopentyl methine), 2.95 (d, 2H, J=6.4, ArCH₂), 2.33 (t, 2H, J=7.4, glu 4-CH₂), 2.00-1.48 (m, 12H, glu 3-CH₂, NH₂, cyclopentyl CH₂), 1.39 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu).

EXAMPLE 192 (S)-tert-Butyl 3-(3-cyclopentyl-4-hydroxyphenyl)-2-((5-O-tert-butyl-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)oxy)propionate

According to example 154, 0.414 g of (S)-tert-butyl 2-((5-O-tert-butyl-L-glutam-1-yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate was coupled with 0.32 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate using 0.19 mL of DECP, 0.23 mL of Et₃ N, and 25 mL of DMF. The crude product was subjected to flash chromatography twice on silica gel (14:14:1 CH₂ Cl₂ :acetone:MeOH; 95:5 91:9 CH₂ Cl₂ :MeOH) to afford 0.46 g (69%) of (S)-tert-butyl 3-(3-cyclopentyl-4-hydroxyphenyl)-2-((5-O-tert-butyl-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)oxy)propionate as a yellow powder, m.p. 135-137° C.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 9.07 (s, 1H, OH), 8.55 (s, 1H, pteridinyl 7-CH), 8.30 (d, 1H, J=7.6, amide NH), 7.71 (d, 2H, J=8.6, aromatic CH), 7.64 (br s, 1H, NH₂), 7.42 (br s, 1H, NH₂), 6.97 (d, 1H, J=1.6, aromatic CH), 6.80 (m, 3H, aromatic CH), 6.59 (m, 3H, aromatic CH, NH₂), 4.92 (t, 1H, J=6.1, propionate methine), 4.77 (s, 2H, pteridinyl-CH₂), 4.49 (m, 1H, glu methine), 3.19 (s, 3H, N--CH₃), 3.12 (m, 1H, cyclopentyl methine), 2.93 (m, 2H, ArCH₂), 2.33 (t, 2H, J=7.2, glu 4-CH₂), 2.08 (m, 1H, glu 3-CH₂), 1.87 (m, 3H, glu 3-CH₂, cyclopentyl CH₂), 1.77-1.40 (m, 6H, cyclopentyl CH₂),1.36 (s, 9H, t-Bu), 1.25 (s, 9H, t-Bu).

EXAMPLE 193 (S)-3-(3-Cyclopentyl-4-hydroxyphenyl)-2-((N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)oxy)propionic acid

According to example 50, 0.43 g of (S)-tert-butyl 3-(3-cyclopentyl-4-hydroxyphenyl)-2-((5-O-tert-butyl-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)oxy)propionate was treated with gaseous HCl in 50 mL of CH₃ NO₂ for 45 min. The yellow suspension was concentrated in vacuo to dryness. The product was suspended in 25 mL of water and was treated with sufficient 1N aqueous NaOH to give complete solution. The solution was filtered and the pH adjusted to 5.5 by addition of 1N HCl. A yellow precipitate resulted which was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, frozen and lyophilized to afford 0.345 g (88%) of (S)-3-(3-cyclopentyl-4-hydroxyphenyl)-2-((N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl)oxy)propionic acid as a yellow-orange powder, m.p. 165° C. (dec).

HPLC: one major peak on C18, k'=2.02; 3.6% of methotrexate was detected at k'=0.09; 70:30:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆) δ 12.40-11.90 (br s, COOH), 9.03 (br s, 1H, OH), 8.57 (s, 1H, pteridinyl 7-CH), 8.27 (d, 1H, J=7.6, amide NH), 7.86 (br s, 1H, NH₂), 7.70 (d, 2H, J=8.9, aromatic CH), 7.65 (br s, 1H, NH₂), 6.97 (d, 1H, J=1.6, aromatic CH), 6.80 (m, 5H, NH₂, aromatic CH), 6.61 (d, 1H, J=8.2, aromatic CH), 4.96 (t, 1H, J=5.9, methine), 4.78 (s, 2H, pteridinyl-CH₂), 4.49 (m, 1H, methine), 3.19 (s, 3H, N--CH₃), 3.16 (m, 1H, cyclopentyl methine), 2.94 (d, 2H, J=5.8, ArCH₂), 2.34 (t, 2H, J=7.5, glu 4-CH₂), 2.10 (m, 1H, glu 3-CH₂), 1.87 (m, 3H, glu 3-CH₂, cyclopentyl CH₂), 1.78-1.40 (m, 6H, cyclopentyl CH₂).

Elemental Analysis: Calcd. for C₃₄ H₃₈ N₈ O₈.2.1 H₂ O (MW 724.56): C, 56.36; H, 5.87; N, 15.47. Found: C, 56.13; H, 5.59; N, 15.46.

Mass Spectrum: (Ion Spray) 687 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 259.2 (23100), 307.3 (23300), 372.4 (7190). λ_(min) (ε) 242.0 (14300), 272.9 (16900), 343.6 (5460). sh (e) 219.8 (26400), 280.7 (19900).

EXAMPLE 194 Methyl 3-tert-butyl-4-hydroxybenzoate

To a 500 mL 3-necked flask equipped with a magnetic stirrer, a nitrogen inlet, and a condenser were added 10.0 g of 3-tert-butyl-4-hydroxybenzoic acid (ICN Biomedicals. Inc., Aurora, Ohio 44202), and 200 mL of MeOH. The solution was acidified by bubbling HCl gas through for 5 min. and was then heated at reflux for 5 h. The solution was cooled to RT and concentrated in vacuo to dryness. The resulting solid was dissolved in EtOAc. The solution was washed three times with 5% aqueous NaHCO₃, dried over anhydrous MgSO₄ and concentrated to dryness to afford 9.65 g (90%) of methyl-3-tert-butyl-4-hydroxybenzoate as a white crystalline solid, m.p. 144-145° C.

EXAMPLE 195 Methyl 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzoate

According to example 183, 5.00 g of methyl-3-tert-butyl-4-hydroxybenzoate was silylated using 4.34 g of tert-butyldimethylsilyl chloride, 3.59 g of imidazole, and 70 mL of anhydrous DMF. The crude product was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 6.1 g (79%) of methyl 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzoate as a clear liquid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.89 (d, 1H, J=2.3, aromatic CH, 7.76 (dd; 1H; J=8.5, 2.3; aromatic CH), 6.98 (d, 1H, J=8.4, aromatic CH), 3.82 (s, 3H, CO₂ CH₃), 1.37 (s, 9H, t-Bu), 1.03 (s, 9H, Si-t-Bu), 0.37 (s, 6H, SiMe₂).

EXAMPLE 196 3-tert-Butyl-4-((tert-butyldimethylsilyl)oxy)benzyl alcohol

According to example 184, 5.00 g of methyl 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzoate was reduced using 31.8 mL of 1M diisobutylaluminum hydride/toluene and 50 mL of anhydrous toluene. Analysis of the reaction mixture by tlc after 30 min indicated some remaining starting material. To ensure complete reduction, an additional 5 mL of 1M diisobutylaluminum hydride/toluene was added and the reaction was allowed to proceed for an additional 5 min. Work-up in the usual manner afforded 4.11 g (90%) of 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl alcohol as a cloudy oil. The product was used without further purification.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 7.20 (d, 1H, J=2.0, aromatic CH), 7.04 (dd; 1H; J=8.2, 2.1; aromatic CH), 6.80 (d, 1H, J=8.0, aromatic CH), 5.01 (t, 1H, J=5.7, OH), 4.39 (d, 2H, ArCH₂ O), 1.36 (s, 9H, t-Bu), 1.02 (s, 9H, Si-t-Bu), 0.32 (s, 6H, SiMe₂).

EXAMPLE 197 3-tert-Butyl-4-((tert-butyldimethylsilyl)oxy)benzyl bromide

A 500 mL round-bottomed flask equipped with a magnetic stirrer was charged with 3.6 g of 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl alcohol and 25 mL of anhydrous CH₂ Cl₂. The solution was cooled in an ice water bath and was treated with 5.68 g of dibromotriphenylphosphorane (Aldrich). The reaction mixture was warmed to RT, stirred for 2.5 h and then diluted with 100 mL of CH₂ Cl₂. The resulting solution was washed with 5% aqueous NaHCO₃ (4×60 mL), dried over anhydrous MgSO₄ and concentrated to give a white solid. The crude product was purified by flash chromatography on silica gel (95:5 hexane:EtOAc) to afford 3.86 g (88%) of 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl bromide as a white crystalline solid, m.p. 54-55° C.

EXAMPLE 198 tert-Butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl)-4-morpholinecarboxylate

According to example 142, 3.82 g of tert-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate was alkylated with 3.86 g of 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl bromide in 80 mL of anhydrous THF using 11.34 mL of 1 M sodium bis(trimethylsilyl)amide in THF. The crude product was subjected to flash chromatography on silica gel (85:15 hexane:EtOAc) to afford 4.85 g (71%) of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl)-4-morpholinecarboxylate as a white foam, m.p. 75-83° C.

EXAMPLE 199 (2R, 3S, 5S)-6-Oxo-2,3-diphenyl-5-(3-tert-butyl-4-hydroxybenzyl)morpholine

To a 250 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer were added 4.51 g of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl)-4-morpholinecarboxylate and 50 mL of anhydrous THF. The solution was cooled in an ice water bath, treated with 7.51 mL of 1M nBu₄ NF/THF (Aldrich), and then allowed to warm to RT with stirring under nitrogen. After 1.5 h, tlc (SiO₂, 85:15 hexane:EtOAc) indicated no remaining starting material and a single new component at R_(f) =0.52. The solution was diluted with 125 mL of EtOAc, washed with water (4×100 mL), dried over MgSO₄, and concentrated to give a thick, clear oil. Mass spectral analysis (APCI) indicated the desired intermediate with m/e=538 (M+H+Na)⁺. The oil was dissolved in 50 mL of CH₂ Cl₂ and the solution treated with 15 mL of TFA. After stirring for 40 min, tlc (SiO₂, 65:35 hexane:EtOAc) indicated a single new component at R_(f) =0.36. The solution was neutralized by addition of saturated aqueous NaHCO₃. The mixture was transferred to a separatory funnel and the layers separated. The CH₂ Cl₂ solution was washed with additional saturated NaHCO₃ (3×50 mL), dried over MgSO₄ and concentrated in vacuo to give a clear foam. The crude product was purified by flash chromatography (SiO₂, 7:3 hexane:EtOAc) to afford 2.66 g (89%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-hydroxybenzyl)morpholine as a white foam, m.p. 76-80° C.

EXAMPLE 200 3-tert-Butyl-L-tyrosine

To a 300 mL Parr bottle were added 0.73 g of PdCl₂ and 20 mL of 1:1 THF:EtOH. To this was added a solution of 2.43 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-hydroxybenzyl)morpholine in 80 mL of 1:1 THF:EtOH. The mixture was deoxygenated by bubbling nitrogen through for 10 min and was hydrogenated at 45 psi for 18 h. The vessel was purged with nitrogen, catalyst removed by filtration, and the filtrate concentrated in vacuo. The residue was partitioned between water and hexane. The aqueous solution was washed with four additional 50 mL portions of hexane, subjected to brief rotary evaporation to remove residual hexane, frozen and lyophilized to afford 1.47 g (92%) of 3-tert-butyl-L-tyrosine.HCl as a tan hygroscopic solid.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.40 (br s, 1H, OH), 8.36 (br s, 3H, NH₃ ⁺), 7.04 (s, 1H, aromatic CH), 6.91 (d, 1H, J=8.2, aromatic CH), 6.78 (d, 1H, J=8.2, aromatic CH), 4.05 (m, 1H, methine), 3.02 (d, 2H, J=5.6, ArCH₂), 1.34 (s, 9H, t-Bu).

EXAMPLE 201 tert-Butyl 3-tert-butyl-L-tyrosinate

To a 250 mL round bottomed flask equipped with a magnetic stirrer were added 30 mL of isobutylene (condensed at -78° C.), 1.30 g of 3-tert-butyl-L-tyrosine.HCl, 60 mL of 1,4-dioxane, and 0.5 mL of concentrated H₂ SO₂. The flask was sealed with a rubber septum and wired shut. After stirring at RT for 18 h, the flask was vented and the contents poured into a solution of 10 g of NaHCO₃ in 150 mL of water. The mixture was subjected to rotary evaporation to remove isobutylene. An emulsion resulted which was extracted with EtOAc (4×40 mL). The combined extracts were washed with 5% aqueous NaHCO₃ (3×50 mL), dried over anhydrous MgSO₄, and concentrated to give a light yellow foam. Analysis of the product by tlc (SiO₂, 95:5 CH₂ Cl₂ :MeOH) indicated major and minor components at R_(f) =0.45 and 0.49 respectively. The R_(f) =0.45 product was isolated by flash chromatography on silica gel (95:5 CH₂ Cl₂ :MeOH) to afford 0.61 g (44%) of tert-butyl-3-tert-butyl-L-tyrosinate as a white foam, m.p. 43-52° C.

EXAMPLE 202 tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate

According to example 145, 0.57 g of tert-butyl 3-tert-butyl-L-tyrosinate was coupled with 0.66 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.39 g of EDC in 20 mL of CH₂ Cl₂ for 4 h. The crude product was purified by flash chromatography on silica gel (65:35 hexane:EtOAc) to afford 0.99 g (83%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate as a white foam, m.p. 62-66° C.

EXAMPLE 203 tert-Butyl 5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate

According to example 48, 0.93 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate was hydrogenolyzed using 0.15 g of 10% Pd(C) in 70 mL of MeOH for 3.5 h. The crude product was subjected to flash chromatography on silica gel (EtOAc) to afford 0.63 g (87%) of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate as a thick transparent oil.

¹ H-NMR: (200 MHz, DMSO-d₆) δ 9.17 (s, 1H, OH), 8.11 (d, 1H, J=7.8, NH), 6.96 (d, 1H, J=1.7, aromatic CH), 6.84 (dd; 1H; J=8.1, 1.9; aromatic CH), 6.68 (d, 1H, J=8.0, aromatic CH), 4.33 (m,1H, methine), 3.14 (m, 1H, methine), 2.84 (d, 2H, J=7.0, ArCH₂), 2.23 (t, 2H, J=7.9, glu 4-CH₂), 1.88-1.46 (m, 4H, glu 3-CH₂, NH₂), 1.40 (s, 9H, t-Bu), 1.33 (s, 18H, t-Bu's).

EXAMPLE 204 tert-Butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate

According to example 154, 0.320 g of tert-butyl 5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate was coupled with 0.254 g of 4-(N-(2,4-diamino-6-pteridinylmethyl-N-methylamino)benzoic acid hemihydrochloride dihydrate using 0.15 mL of DECP, 0.19 mL of Et₃ N, and 20 mL of DMF. The crude product was purified by flash chromatography on silica gel (7:7:1 CH₂ Cl₂ :acetone:MeOH) to afford 0.32 g (61%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate as a yellow powder, m.p. 125° C. (dec).

EXAMPLE 205 N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-tyrosine

According to example 50, 0.287 g of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-1-yl-3-tert-butyl-L-tyrosinate was treated with gaseous HCl in 55 mL of CH₃ NO₂ for 1 h. The suspension was concentrated in vacuo to give a yellow powder. This material was suspended in 30 mL of water and treated with sufficient 1N aqueous NaOH to give complete solution. The yellow solution was filtered and acidified to pH=5.5 by addition of 1N aqueous HCl. The resulting yellow precipitate was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, frozen and lyophilized to afford 0.178 g (68%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-tyrosine as a yellow powder, m.p. 185° C. (dec).

HPLC: one peak on C18, K'=3.14, 75:25:0.1 H₂ O:MeCN:TFA, flow rate=1 mL/min.

¹ H-NMR: (300 MHz, DMSO-d₆)δ 12.80-11.80 (br m), 9.09 (s, 1H, OH), 8.55 (s, 1H, pteridinyl 7-CH), 8.01 (d, 1H, J=7.9, amide NH), 7.97 (d, 1H, J=8.1, amide NH), 7.78 (br s, 1H, NH₂), 7.68 (d, 2H, J=8.9, aromatic CH), 7.57 (br s, 1H, NH₂), 6.93 (d, 1H, J=1.6, aromatic CH), 6.85-6.64 (m, 5H, aromatic CH, NH₂), 6.60 (d, 1H, J=8.2, aromatic CH), 4.77 (s, 2H, pteridinyl-CH₂), 4.42 (m, 1H, methine), 4.29 (m, 1H, methine), 3.19 (s, 3H, N-CH₃), 2.83 (m, 2H, ArCH₂), 2.23 (t, 2H, J=7.8, glu 4-CH₂), 1.90 (m, 2H, glu 3-CH₂), 1.25 (s, 9H, t-Bu).

Elemental Analysis: Calcd. for C₃₃ H₃₉ N₉ O₇ ·2.3 H₂ O (MW 715.16): C, 55.42; H, 6.14; N, 17.63. Found: C, 55.58; H, 5.97; N, 17.47.

Mass Spectrum: (Ion Spray) 674 (M+H)⁺.

UV Spectrum: (pH 7 Buffer) λ_(max) (ε) 259.4 (23700), 307.4 (23500), 372.5 (7330). λ_(min) (ε) 242.8 (14900), 273.0 (17000), 344.7 (5660). sh (e) 222.1 (26400), 287.2 (19600).

EXAMPLE 206 Sequencing of Human Carboxypeptidase A1 cDNA

A Hind III - Sal I cDNA fragment encoding the rat preprocarboxypeptidase A1 (CPA1) (Gardell et al., Nature, 317:551-555, 1985) was isolated from pMP36 provided by Dr. M. Phillips (UCSF). The 1.2 kb Hind III - Sal I rat CPA1 cDNA was radiolabeled with [³² P]dCTP and Prime-it kit (Stratagene) and then used to screen a lambda gt11 human pancreas cDNA library (Clontech) according to the method of Grunstein and Hogness (Proc. Nat. Acad. Sci 72: 5016-5020, 1975). Purified isolated plaques that hybridized to the rate CPA1 cDNA probe were obtained after three rounds of screening.

Lambda DNA from purified plaques was prepared from overnight growth liquid cultures using Qiagen columns and according to the manufacturer's protocol (Qiagen). Human CPA1 cDNA inserts were liberated from lambda DNA by digestion with EcoRI and purification of the cDNA inserts by low-melting agarose gel electrophoresis. The EcoRI cDNA inserts were cloned into the EcoRI site of pGEM 7z(+) (Promega) and denoted as pHCPA plasmids. Following overnight growth in liquid culture, plasmid pHCPA DNA was prepared using Qiagen columns.

DNA sequencing of isolated HCPA1 lambda plaques and of plasmids containing HCPA1 cDNA was performed with [³⁵ S]dATP and the fmol sequencing system (Promega). Oligonucleotide primers that flanked the cloning site of lambda gt11 or the multiple cloning sites in pGEM 7(+) and the TA vector (Invitrogen) were used initially to determined cDNA sequence. Oligonucleotide primers that corresponded to regions of the determined sequence were synthesized which permitted the entire cDNA sequence of both strands of HCPA1 to be determined. (Seq ID NO: 1)

EXAMPLE 207 Cloning of Human Carboxypeptidase A2

Human carboxypeptidase A2 (CPA2) was cloned from a human pancrease cDNA library constructed in lambda gt11 (Clontech). The probe used as an 1198 base fragment of rat CPA2, This probe was amplified from QUICK-Clone rat pancreas cDNA (Clontech) using PCR. The upstream primer corresponded to the sequence between positions 12-33 in the rate cDNA coding sequence (5'-CCTGTTATTGGCTGCCCTACTT-3')(SEQ ID NO: 5), while the downstream primer was the complement to the sequence between positions 1888-1209 (5'-AAGCCAGGTCTCTTCTGCTGTC-3') (SEQ ID NO: 6) (Gardell, S. J. J. Biol. Chem. 263 17828 (1988)). The standard PCR conditions were a 94° C. preincubation followed by 30 cycles of one minute at 94° C., two mins at 56° C., and three mins at 72° C. The 1198 bp PCR product was extracted with 100 ul chloroform and the upper aqueous phase containing the DNA was passed through a CHROMA SPIN-100 column (Clontech) to remove the primers. The sequence of the rate CPA2 PCR product was confirmed with DNA sequencing using the PCR primers as sequencing primers. Sequencing of the DNA was done with [³² P] dATP using the fmol sequencing kit (Promega).

The rat CPA2 DNA was radiolabelled with [³² P] dCTP and the Prime-it II Random Primer Labelling kit (Stratagene) and then used to screen the human pancreas cDNA library. The library was plated using E. coli strain Y1090 on 150 mm plates (approximately 10,000 plaques/plate) and duplicate nitrocellulose lifts made. The filters were blocked in hybridization solution (50% formamide, 5X SSPE, 50X Denhardt's solution, 0.1% SDS, and 100 ug/ml sonicated salmon sperm DNA) for 3 hours at 42° C. Labelled rate CPA2 PCR product was added and hybridized to the filters overnight at 45° C. The filters were washed for 1 hour at 60° C. in 0.1X SSC, 0.1% SDS and exposed to autoradiography film while moist. One duplicate plaque from each plate was picked and subjected to another round of screening as described above. Positive plaques after the second round of screening were picked and the size of the lambda gt11 insert DNA was determined using PCR. Lambda gt11 forward and reverse sequencing primers (Clontech) that flanked the EcoRI cloning site of lambda gt11 were used as the upstream and downstream PCR primers, respectively. Well-isolated plaques were picked, with a portion suspended in 10 ul water, and heated at 95° C. for 5 min, and the remainder being used to make a phage stock for future screening. The 10 ul containing the plaque DNA was used directly as the template in PCR using the conditions described above.

Amplified insert DNA was visualized on agarose gels and the two plaques giving the largest insert DNA were screened a third time. Lambda DNA from the two plaques was isolated and sequenced in both directions using the fmol kit (Promega) and the lambda gt11 forward and reverse primers (Clontech). One clone contained regions highly homologous to the 5' and 3' regions of rat CPA2 and was chosen for further study.

The insert cDNA was liberated from the lambda DNA by digestion with EcoRI, gel purified, and cloned in the EcoRI site of the pGEM-7Zf(+) vector (Promega). Sequencing was carried out using [^(33P) ] dATP and the fmol kit (Promega) as described above. Oligonucleotide primers that corresponded to regions of the determined sequence were synthesized which permitted the entire cDNA sequence of hCPA2 to be determined. The cDNA was found to contain an open reading frame coding for a protein of 417 amino acids. This protein shares 87% identity with rate CPA2 at the amino acid level and 85% identity at the cDNA level. This protein shares 63% identity with human CPA1 at both the amino acid and nucleotide levels. Thus the protein was classified as human carboxypeptidase CPA1 and CPA2. The amino acid numbering scheme used throughout this document is that of CPA1. All mutations described for CPA 1 and CPA2 refer to this numbering scheme.

EXAMPLE 208 Expression of HCPA1 in Yeast

The size of cDNA inserts within isolated HCPA1 lambda plaques was determined using oligonucleotide primers that flanked the EcoR1 cloning site of lambda gt11 and polymerase chain reaction (PCR). Single isolated, plaques were picked, suspended in 200 μl water and heated at 95° C. for 5 minutes. Ten μl of each suspension was used for PCR amplification according to manufacturer's instructions (Perkin Elmer/Cetus). The standard PCR reaction conditions consisted of 1 minute at 95° C., 0.5 minute at 50° C., and 3 minutes at 72° C. The reaction products were visualized following agarose gel electrophoresis.

CPA-11 5'-gCT gAA gCT TCg gAg gAC TTT-3' (SEQ ID NO: 15)

CPA-12 5'-TCT TgA CCg CCT ggA TgC Tg-3' (SEQ ID NO: 16)

The express of HCPA1 in S. cerevisae was based on the strategy employed by Gardell et al (Nature, 317; 551-555, 1985). Two oligonucleotide primers, CPA 11 and 12, were used to amplify a 200 bp fragment from pHCPA by PCR as described above. The amplified fragment contained a new 5' Hind III site created by CPA 11 that would allow ligation in frame with the alpha factor leader of pMP36 as described by Phillips et al (J. Biol. Chem. 265; 20692-20698, 1990). The PCR fragment was ligated into the TA vector (Invitrogen) and the entire DNA sequence of the fragment determined as described earlier. A colony that contained the desired 5' alteration was selected for further use, TAHCPAexp.

Plasmid pHCPA was restricted with Aat II and EcoR1 to liberate a 1.1 kb HCPA1 fragment. This fragment was purified following low melting point agarose gel electrophoresis and ligated into TAHCPAexp that had been restricted with AatII amd EcoR1 to yield the plasmid TAHCPA. The TAHCPA was digested with EcoR1, treated with T4 DNA polymerase, and Sal I linkers were added and ligated to the 3' end of HCPA1 as described by Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989). TAHCPA that now contained 5' Hind III and 3' Sal I site was restricted with Hind III and Sal I to liberate a 1.2 kb HCPA1 cDNA fragment. This fragment was ligated together with the large Hind III fragment and the small Hind III-SalI fragment of pMP36 to produce pMP36 HCPA1.

Yeast expression of the cloned human enzyme is based on the method of Phillips, M. et al. J. Biol. Chem. 265, 20692 (1990). Plasmid pMP36 containing HCPA1 was restricted sequentially with BAMH1, SAL1 and SSP1 with intervening purification by either phenol extractions or use of Promega Magic Mini Columns with manufacturer supplied procedures. Alternatively, the One-Phor-All buffer system (Pharmacia) can be used in a concurrent restriction by all three enzymes.

Following SSP1 restriction, the mixture was separated on a 1% low melting gel giving 5 distinct bands. The band of interest at about 2.8 kb (the second from top) was excised from the gel and purified using a Schleicher and Schuell Elutip-D column with the supplied procedures.

The BAM/SAL/SSP fragment was then ligated into pBS24.1 shuttle vector (produced from pBS24.1-AGC3 kindly provided by Dr M Phillips, UCSF with BAMH1/SAL1 restriction) overnight at 16° C. using the T4 ligase system (Gibco BRL). The ligated vector was precipitated with ammonium acetate and ethanol at -80° C. for 4 hrs and resuspended in 10 μl TE.

4 μl of the vector was electroporated into 40 μl of DH5alpha E. coli using a Bio RAd Gene Pulser at 2000 volts with a time constant around 4. One ml of Miller Hinton broth was added and the sample incubated at 37° C. for one hour. Three 100 μl samples of each was plated out onto ampicillin containing LB plates.

The resultant colonies were replated on fresh plates. 100 ml volumes of Mueller-Hinton/ampicillin broth were innoculated with colonies and incubated overnight at 37° C. The plasmid was extracted using the Qiagen Maxi extraction procedure.

Approximately 500 to 2000 ng of the PBS24.1 plasmid DNA extracted above was electroporated into 40 μl of competent DLM101 alpha yeast cells. One ml of sorbitol was added immediately after electroporation to rescue the cells. 100 μl samples were plated out on dishes of YNB (yeast nitrogen broth) lacking uracil which were incubated at 30° C. Positive clones were picked. Transformants were grown in minimal synthetic medium (Wickersham, L. J., U.S. Dept. of Agric. Tech. Bull. No. 1029 (1951), lacking leucine, for 48 hrs. This culture was then used to seed a 350 liter fermenter.

EXAMPLE 209 Purification of HCPA1 or its Mutants From Yeast

A 350 l culture of proCPA-producing S. cerevisiae (T268G/p2619) was grown in a 500 l New Brunswick fermentor for 65 h at 30° C. with 175 lpm air flow, 5 psig head pressure and 150 rpm agitation. The medium contained 1% yeast extract (Difco), 2% tryptone (Difco), 1% glucose, 20 mM KPO₄, pH 7.5, 50 μg/ml ampicillin and 0.007% (v/v) Mazu DF 60P antifoaming agent (Mazei Chemicals, Inc.). The final OD₆₀₀ was 14. After cooling to 15-20° C. the cells were removed from the medium by cross-flowfiltration on a Sartorius Sartocon II system equipped with 7 0.6 m² polyolefin filter units. The protein in the medium was concentrated with 10K MWCO cross-flow filters and the fluid exchanged by diafiltration with 20 mM Tris-Cl. pH 8, 0.1 mM Zn-acetate (buffer A). All subsequent buffers contained 0.1 mM Zn-acetate. Ammonium sulfate (AS) was added to 0.5M to the 10K concentrate and it was loaded onto a 250 ml radial flow Phenyl-sepharose column equilibrated in buffer A with 0.5 M AS. The proCPA was eluted with a 1.5 l 0.5-0 M AS gradient followed by a buffer A wash. The Phenyl-Sepharose procedure was repeated with the breakthrough material to recover unbound proCPA from the first run. Fractions containing proCPA were pooled and concentrated and dialyzed on a Sartorius 10K Easy Flow filter (0.2 m²) into buffer A. This was loaded onto a Hi Load 26/10 Q-Sepharose High Performance (Pharmacia) column (53 ml) and eluted with a 1 liter 0-0.5M NaCl gradient in buffer A. The proCPA eluted between 120-220 mM NaCl with the peak tube at ˜150 mM. Fractions containing proCPA were pooled and the proCPA was cleaved to mature CPA with trypsin (2 μg/ml) at 37° C. for 1 h. PMSF was added (0.5 mM) and the pool was dialyzed in buffer A. The Q-Sepharose HP step was repeated and the activated CPA eluted between 80-140 mM with the peak tube at 120 mM. Previous IEF experiments have shown the proCPA has a pI=4.75 while the mature CPA has a pI=6.2. The CPA fractions were pooled and concentrated with 10K Centripreps (Amicon) to 15 ml and this was passed through a HiLoad 26/60 Superdex 75 (Pharmacia) column in 3 runs (5 ml each). The elution buffer was buffer A with 150 mM NaCl. The CPA fractions were pooled, concentrated with 10K Centripreps and exchanged with Sigma PBS containing 0.1 mM Zn-acetate. The final volume (15 ml) was passed through a 0.2 μ syringe filter and stored at 4° C. The protein was 24.5 mg/ml by the Lowry assay using BSA standards. The specific activity for a typical preparation of the mutant with Gly at 268 was 163 u/mg. Total protein=368 mg.

EXAMPLE 210 Expression of HCPA2 in Yeast

The expression of HCPA2 in S. cerevisiae was employed the same strategy described for HCPA1 (Example 208) with the following exceptions: 1) the PCR primers used to introduce the new 5' Hind III site were:

YEAST 1: 5'-gCA gAA gCT TCA gAA ACg TTT gTg ggA gAT CAA gTTCTT gAg ATT gTA CC-3' (SEQ ID NO: 19)

YEAST 2: 5'-CTC TTT gTC CAA CAg gAC CTg-3' (SEQ ID NO: 20)

2) a Hinc II site rather than Aat II was used to subclone the mutagenic PCR fragment to the 5' end of the HCPA2 cDNA, and 3) the cloned PCR product was cut from the TA vector and subcloned into the pGEM-7Zf(+) vector containing the HCPA2 cDNA. PCR was carried out with the YEAST 1 and 2 primers as described for HCPA1. The mutagenic PCR product was cloned into the TA vector and the sequence confirmed. This fragment was cut from the TA vector using Hind III and Hinc II and cloned into the corresponding sites of pHCPA2 and the resulting construct designated pHCPA2-Y. The unique 3' Eco RI site of pHCPA2-Y was changed to a Sal I site using linkers as described above with the resulting construct designated pHCPA2-YSal I. The 1.2 kb Hind III/Sal I HCPA2 fragment derived from pHCPA2-Sal I was excised from the vector and ligated together with the large Hind III/Sal I fragment of pMP36 (Phillips, M. et al, J. Biol. Chem., 265, 20692-20698 (1990)) to produce pMP36HCPA2.

Yeast expression of clone HCPA2 was as described above for HCPA1 (Example 208). Human CPA2 has two internal Bam HI sites not present in human CPA1 so partial digestion was needed for the final subcloning step. pMP36HCPA2 was digested to completion with Sal I and partially digested with Bam HI (as described above) to yield the 2.7 kb fragment containing the alcohol dehydrogenase/GADPH promoter and regulatory region (A/G promoter) attached to human CPA2 fused in frame to the yeast α factor leader. This fragment was ligated into the Sal I/Hind III site of pBS24.1 forming pBSHCPA2. The construct was amplified in DH5α and used to electroporate yeast as described above (Example 208).

EXAMPLE 211 Purification of HCPA2 or it Mutants From Yeast

An overnight culture (grown in Leu⁻ medium) of S. cerevisiae expressing proCPA2 as used to start 2 l of culture in 1% yeast extract (Difco), 2% tryptone (Difco), 1% glucose, 20 mM KPO₄, pH 7.5, and 50 μg/ml ampicillin. The culture was grown for 48 hrs at 30° C. and the supernatant cleared by centrifugation at 6000 X g for 10 min at 4° C. The protein in the supernatant was concentrated on an Amicon PM-10 Ultrafiltration membrane to 114 ml. The concentrated supernatant was made to 20 mM Tris-HCl (pH 8.0), 0.1 mM Zn-acetate and 0.5 M ammonium sulfate and loaded onto a 1.8×20 cm Phenyl-Sepharose column (Pharmacia) equilibrated in buffer A with 0.5 M ammonium sulfate. The protein was eluted with a 120 ml linear gradient (1 ml/min) from buffer B (20 mM Tris-HCl pH 7.5, 0.1 mM Zn-acetate) with 0.5 M ammonium sulfate to buffer B with 20% (v/v) ethanol. Fractions containing carboxypeptidase activity were pooled and dialyzed against buffer B. The dialyzed pool was loaded onto an HR 5/5 (1 ml) Mono Q column (Pharmacia) and eluted with a 30 ml linear gradient (1 ml/min) from 0-0.5 M NaCl in buffer B. Fractions containing carboxypeptidase activity were pooled and dialyzed against buffer B. The pooled proCPA2 was activated with trypsin (10 μg/ml) at 37° C. for 1 hr and the trypsin inactivated with PMSF added to 0.5 mM. The mature CPA 2 was dialyzed against buffer B and again loaded onto the 1 ml Mono Q column (Pharmacia) and eluted as above. Fractions containing carboxypeptidase activity were pooled, dialyzed against buffer B, and concentrated 10 fold in an Amicon Ultrafiltration cell using a PM-10 membrane. The concentrated mature CPA2 was loaded onto an HR 10/30 Superose 12 column (25 ml) and eluted isocratically (0.4 ml/min.) in phosphate buffered saline (10 mM Na₂ HPO₄, 0.15 M NaCl, pH 7.2). For wild type HCPA2 the protein was 0.65 mg/ml (4.13 mg total) and for the 268 Gly mutation the concentration was 0.3 mg/ml (4.47 mg total) as determined with the Micro BCA Assay (Pierce Chemical Co.) using BSA standards.

EXAMPLE 212 Expression of Mutant HCPA2

The A250G and T268G mutations of HCPA2 were constructed as described above (Example 216) for mutant forms of HCPA1 using the T7-Gen In Vitro Mutagenesis Kit (United States Biochemical, No. 74500). The mutagenic oligonucleotide primers (Oligos Etc.) to mutate the A250 (gCC) and T268 (ACC) to Gly (ggC) are shown below:

A250G 5'-TCC TCC ACT gCC TTg gTA gAT-3' Gly=ggC (SEQ ID NO: 21)

T268G 5'-CAg TTC AAA gCC AAA TgA gTA-3' Gly=ggC (SEQ ID NO: 22)

Mutagenic codons are underlined.

Using these oligonucleotide primers the following mutant forms of HCPA2 were produced:

1. A250G

2. T268G

Each of the above mutagenized HCPA2 cassettes was sequenced to verify that only the desired DNA mutations were produced. The mutagenized cassettes were subcloned into pMP36HCPA2 that could further be processed for expression in S. cerevisiae using methods described in Example 210.

EXAMPLE 213 Synthesis of a Chemical Conjugate Between Campath-1-H or ING-1 and Human Carboxypeptidase A Mutants

2 mg of mutant carboxypeptidase A was combined with 0.15 mg of Sulfo-SMCC (Pierce Chemical Co) in 475 μl of Dulbecco's Phosphate Buffered Saline (PBS). The resulting solution was stirred for 45 minutes at 25° C. The modified enzyme was purified through a 1×13 cm G-25 medium column equilibrated with PBS. Purified product can be stored at 4° C. for 24 hrs.

Maleimide content was determined by combining 0.5 ml of 6.3 μM modified enzyme with 6 μl of 1 mM mercaptoethanolamine. After 30 minutes, 20 μl of 4 mg/ml Ellman's reagent was added, and after another 20 minutes absorbance at 412 nm was determined. Based upon a molar absorptivity of 13.6 mM⁻¹, the purified product was found to contain 1 maleimide per enzyme molecule. The modification had no effect upon enzyme activity.

The antibody was modified with 2-iminothiolane (Pierce Chemical Co). 0.35 mg of antibody solution was combined with 0.055 mg of 2-iminothiolane in 0.1 M triethanolamine-HCl, 2 mM EDTA, pH 8.0 under anaerobic conditions, reacted with stirring for 1 hr and 45 mins. The modified antibody was purified through a 1×13 cm G-25 medium column equilibrated with 0.02 M sodium acetate, 0.1 M NaCl, pH 5.8, bubbled with and maintained under a He atmosphere. The purified modified antibody solution was collected directly into the solution of modified carboxypeptidase A. The resulting solution was adjusted to pH 7.4 with NaOH, made anaerobic, and allowed to react, with stirring, at 4° C. for 18 hours. (Optionally free maleimide groups may be removed by reacting the solution with 0.3 mM mercaptoethanolamine at room temperature for one hour). The solution concentrated to 1 ml. The resulting concentrated conjugate was purified from aggregates, unreacted enzyme and small molecules by chromatography on Superose 12 HR 10/30. The enzyme specific activity of the conjugate was found to be appropriate for a one to one conjugate with antibody. Antibody (Campath-IH® or ING-1) % immunoreactivity and antigen binding affinity of the conjugate were both found to be high.

EXAMPLE 214

The enzyme was modified by the protocol described for Example 213 above, while the antibody was modified with dithiothreitol. 0.7 mg of antibody solution was combined with 0.035 μmol of dithiothreitol in 350 μl of 0.1 M triethanolamine-HCl, 2 mM EDTA, pH 8.0 under anaerobic conditions and reacted with stirring for 1 hr. The modified antibody was purified through a 1×13 cm G-25 medium column equilibrated with 0.02 M sodium acetate, 0.1 M NaCl, pH 5.8, bubbled with and maintained under a He atmosphere. The purified antibody solution was collected directly into the solution of NaOH, made anaerobic, and allowed to react, with stirring, at 24° C. for 30 min and at 4° C. for 18 hrs. Then the solution was concentrated to 1 ml. The resulting concentrated conjugate was purified from aggregates, unreacted enzyme and small molecules by chromatography on Superose 12 HR 10/30. The enzyme specific activity of the conjugate was found to be appropriate for a one to one conjugate with antibody. Antibody % immunoreactivity and antigen binding affinity were both found to be high.

EXAMPLE 215

The enzyme was modified by the protocol described for Example 4 above, while the antibody was modified with SATA (Pierce Chemical Co). 0.225 mg of antibody solution was combined with 0.003 mg of SATA in a total volume of 150 μl of Dulbecco's Phosphate Buffered Saline (PBS) and allowed to react with stirring for 45 mins. the modified antibody was purified through a 1×13 cm G-25 medium column equilibrated with PBS. The purified modified antibody solution was mixed with the modified enzyme and made anaerobic, 300 μl of anaerobic 0.5 M NH₂ OH was added and the solution allowed to react, with stirring, at 25° C. for 2 hrs followed by 4° C. for 18 hrs. Free maleimide groups were removed by reacting the solution with 0.3 mM mercaptoethanolamine at room temperature for one hour, and the solution was then concentrated to 1 ml. The resulting concentrated conjugate was purified from aggregates, unreacted enzyme and small molecules by chromatography on Superose 12 IIR 10/30. The enzyme specific activity of the conjugate was found to be appropriate for a one to one conjugate with antibody. Antibody antigen binding affinity was found to be high.

EXAMPLE 216 Expression of Mutant HCPA1

pMP36 containing wild type (WT) proHCPA1 cDNA (as a fusion with yeast alpha-factor leader described above, see FIG. 7) was restricted with Nco I and Sal I to liberate a 481 bp cDNA fragment. This fragment begins a nucleotide 893, proceeds to the very 3' end of HCPA1 cDNA and encodes amino acids 186-309 of mature HCPA1. The HCPA1 Nco I-Sal I fragment was ligated into the Nco I and Sal I cloning sites of pGEM5zf(-) (Promega) to generate pHCPAINS (FIG. 3). pHCPAINS was then restricted with Sph I and Sal I to liberate the HCPA1 Nco I-Sal I fragment and additional (9 bp) pGEM5zf(-) sequence. This Sph I-Sal I fragment was cloned into M13mp19 (BRL) using its Sph I and Sal I cloning sites to generate M13mp19HCPA1 (FIG. 4).

Single stranded M13mp19HCPA1 DNA was used as template for olignucleotide-directed mutagenesis using the T7-GEN In Vitro Mutagenesis Kit (United States Biochemical, No. 74500). Mutagenic oligonucleotide primers (Oligos Etc) listed below were used to mutate residues I255 (ATT) and T268 (ACC) either separately or in tandem.

1. I255A 5'-ggT CCA gTC AgC AgT gCT TCC-3' Ala=gCT (SEQ ID NO: 7)

2. T268A 5'-gAg CTC gAA ggC gAA ggA gTA-3' Ala=gCC (SEQ ID NO: 8)

3. T268G 5'-gAg CTC gAA gCC gAA ggA gTA-3' Gly=ggC (SEQ ID NO: 9)

Mutagenic codons are underlined.

Using these oligonucleotide primers the following mutant forms of HCPA1 were produced.

1. I255A

2. T268A

3. T268G

4. I255A/T268A

Each of the above mutagenized HCPA1 cassettes was sequenced to verify that only the desired DNA mutations were produced.

pMP36 HCPA1 w.t. was restricted with NcoI to liberate a 1.2 kb fragment that was cloned into the Nco I site of M13mp19HCPA1 mutants. After the correct orientation of the Nco I fragment within M13mp19 proHCPA1 mutants (FIG. 5) was verified the DNAs were restricted with Hind III and Sal I which liberated a 1.2 kb cDNA fragment encoding the entire proHCPA1mutant enzyme. This fragment was then ligated into the Hind III and Sal I sites of pMP36 yielding pMPHCPA1 mutants (FIG. 6) that could then be further processed for expression in S. cerevisae using the methods described in Examples 208 and 209.

EXAMPLE 217 Expression of Mutant HCPA1

Alternatively, the mutations have been conveniently generated in hCPA in the pMP36 plasmid (pMP36 HCPA1) by the unique site elimination (USE) procedure of Clontech Laboratories, Inc. using the manufacturers' reagents and protocols.

In the protocol, elimination of a unique EcoRI in the pMP36 HCPA1 plasmid was used for selection with the oligonucleotide XS5 (TCC CCC GGG CTG CAG GAT ATC GAT (SEQ ID NO: 10) AGC TGG GCT GTG T). The oligonucleotides used to generate three specific mutations in hCPA by this protocol follow:

T 268 H (ACC codon to CAC) with oligonucleotide XT2 (ATC AAG TAC TCC TCC CAG CTC CGG GAC) (SEQ ID NO: 11)

S 253 G (AGC codon to GGT) with oligonucleotide XT3 (TTT ATC AAG CCA GTG GAG GTA TTG ACT GGA CC) (SEQ ID NO: 12)

A 250 G (GCC codon to GGC) with oligonucleotide XT4 (AAG GCA ATT TAT CAA GGC AGT GGA AGC ACT ATT) (SEQ ID NO: 13)

The mutations were isolated using the oligonucleotides listed above and XS5 (TCC CCC GGG CTG CAG GAT ATC GAT AGC TGG GCT GTG T) (SEQ ID NO: 14) which eliminates a unique EcoRI site. Plasmids containing the desired mutations were selected by digesting the wild type pMP36(hCPA) with EcoRI (uncut plasmids transform 10 to 100× more efficiently than linearized plasmid). In addition, the XS5 selection oligonucleotide introduces an EcoRV site which can then be used to screen for mutant plasmids. In the mutant plasmids the EcoRV is unique and can therefore be used as a selection site to construct double mutants with an oligonucleotide than converts the EcoRV site back to an EcoRI site.

The mutant pMP36 HCPA so generated could then be further processed for expression in S. cerevisae using the methods described in Examples 208 and 209.

EXAMPLE 218 Stability and Biodistribution of Prodrugs In Vivo

Mice were dosed i.p. or i.v. bolus with 50 mg/kg of prodrug by administration of a 5 mg/ml solution in PBS, pH 6-8. Animals were sacrificed after 30 min and 2 h and the plasma, and tissues were collected. Plasma was frozen at -70° C. and tissues were snap frozen in liquid nitrogen and stored at -70° C. Duplicate animals were sacrificed for each time point. Plasma was prepared for extraction by 4-fold dilution with ice cold 0.1 N HCl. Tissues were prepared for extraction by homogenization in 5 volumes of ice cold 0.1 N HCl with a Polytron equipped with a PTA 7 generator (Brinkman Instruments, Westbury, N.Y.). Drugs were subsequently extracted by adding 500 μl of -20° C. acetonitrile to 200 μl of the 0.1N HCl homogenate. After a 5 min incubation on ice, samples were centrifuged at 12,400 x g for 15 min. Supernatants were collected and diluted 3.57 fold with PBS to reduce the final acetonitrile concentration to 20%. Diluted supernatants were then analyzed by HPLC as described below. Drug recovery was estimated by spiking control tissue homogenates with either drug or prodrug prior to the acetonitrile extraction step. After correction for recovery of these standards, the levels of drug and prodrug in tissues and the stability of the prodrug in these tissues were calculated using HPLC.

Samples were analyzed on a 4 μm C18 Nova-Pak 3.9×300 mm steel HPLC column equipped with a C18 guard column with UV detection at a flow rate of 1 ml/min.

Samples were injected onto the HPLC column equilibrated with 2% acetic acid, pH 3.0, containing either 5% or 20% acetonitrile depending upon the compound being analyzed. Upon sample injection a 25 or 35 min linear gradient was initiated raising the acetonitrile concentration to 50% acetonitrile. Three compounds (ASP-MTX, o-cyclopentyl-PHE-MTX and m-cyclopentyl-PHE-MTX) were analyzed on a 10 μm C18 uBondapak column (3.9×300 mm) equilibrated with 16% acetonitrile in 5 mM tetrabutylammonium hydrogen sulfate, 10 mM NH₄ H₂ PO₄. The column was then eluted with a 30 min linear gradient of up to 50% acetonitrile.

Results indicated in Table 1. The results show that a compound that is a good substrate for wild type CPA, such as compound #10, is not stable in vivo. This type of compound is not useful for ADEPT. However, compounds which are good substrates for a mutant enzyme but not for the wild type enzyme, such as compounds 8, 9, 11, 12, 13 and 14 and others, are stable in vivo and are as such useful in ADEPT.

The data shown in Table 1 indicate the concentration of prodrug detected over time in minutes as either μM in the case of plasma or nmol/g in the case of tissues. The figures in brackets indicate the stability of the prodrug expressed as the percentage of material (sum of drug and prodrug) observed as intact prodrug.

Except as indicated all experiments were carried out in CD-1 nude mice.

                                      TABLE 1                                      __________________________________________________________________________     Prodrug                                                                             Time                                                                               PLASMA     LIVER      KIDNEY     SPLEEN                               __________________________________________________________________________     1    30  41.5  (99) 770.3                                                                                (98) 175.8                                                                                (98) 23.4 (100)                             259W91 120 4.2 (100) 579.8  (97) 70.3  (97) 1.8 (100)                          2 30 33.0 (100) 42.0  (98) 9.0  (98) ND                                        519W91 90 5.5  (96) 9.1 (100) 1.3  (98) ND                                      180 3.5  (97) 0.8 (100) 0.2 (100) ND                                          2 30 22.1 (100)                                                                519W91 120 0.3  (93)                                                           3 30 96.3  (93) 128.8  (79) 41.7  (82) 76.6  (95)                              552W92 120 5.1  (87) 267.9  (74) 9.2  (74) 12.1  (89)                          3 30 32.4  (93) 135.3  (87) 29.2  (89) 30.9 (100)                              552W92 120 2.8  (48) 371.2  (90) 4.0  (74) 1.5 (100)                           4 30 71.4 (100) 343.4  (99) 94.5 (100) 30.9  (99)                              2484W92 120 1.4  (99) 33.3  (98) 14.5  (99) ND                                 5 30 114.6  (98) 118.2  (81) 42.5  (93) 34.3  (85)                             2749W92 120 9.4  (95) 7.5  (41) 3.0  (96) 32  (81)                             6 30 46.7 (100) 287.8  (97) 93.4  (98) 3.4  (70)                               3347W92 120 2.0 (100) 161.6  (93) 12.6  (98) 0.0  (0)                          7 30 51.4 (100) 171.9 (100) 55.4 (100) 12.4 (100)                              3855W92 120 0.2 (100) 7.5  (89) 3.8 (100) 1.0 (100)                            8 30 28.5  (95) 113.5  (90) 5.6  (84) 1.4  (98)                                250W93 120 1.1  (90) 43.9  (76) 3.2  (81) 1.1 (100)                            9 30 1.1 (100) 54.3  (83) 6.8 (100) 8.4 (100)                                  637W93 120 ND  126.2  (94) 1.3 (100) ND                                        (BALB/C) 30 4.9 (100)                                                          10 30 17.8 (30.2) 335.1  (94.425) 26.9 (100) 19.9  (80.4)                      1311W92 120 1.1  (2.1) 0.6  (7.55) 0.0  ND                                     11 30 222  (97.075) 53.8  (81.35) 7.3  (83.475) 2.7  (95.45)                   3352W93 120 ND  47.8  (73.325) 3.0  (82.3) ND                                  12 30 2.6 (100) 58.6  (91.325) 2.2  (53.433) ND                                1834W93 120 0.7 (100) 39.5  (89.25) 0.2  (21.55) ND                            13 30 3.3 (100) 47.7 (100) 3.7 (100) 0.6 (100)                                 5755U93 120 0.6 (100) 129.0 (100) 0.5 (100) 0.2 (100)                          14 30 1.1 (100) 22.5 (100) 4.6 (100) 2.4 (100)                                 4204W93 120 ND  42.1 (100) ND  ND                                            __________________________________________________________________________             LARGE             SMALL                                                  Prodrug Time INTESTINE CECUM INTESTINE FECES TUMOR                           __________________________________________________________________________     1    30 12.2                                                                                (93)                                                                               6.0  (96)                                                                               48.2                                                                                (83)         6.0  (96)                            259W91 120 1.0 (100) 1.2  (76) 55.6  (68)   1.2  (76)                          2 30 5.9  (93) 2.7  (98)     2.7  (98)                                         519W91 90 0.7  (60) ND      ND                                                  180 1.3  (52) ND      ND                                                      2 30     170.2  (91)                                                           519W91 120     17.6  (47)                                                      3 30 23.4  (87)                                                                552W92 120 1.7  (89)                                                           3 30 14.9  (94)   27.7  (10)                                                   552W92 120 1.1  (87)   1.8  (1)                                                4 30 25.3 (100) 6.7 (100) 188.1  (99)   6.7 (100)                              2484W92 120 45.4 (100) 1.3 (100) 69.1  (98)   1.3 (100)                        5 30 28.4  (93) 4.7  (95) 32.1  (40)   4.7  (95)                               2749W92 120 1.6  (8) 2.9  (88) 1.6  (2)   2.9  (88)                            6 30 5.7 (100) 7.5  (82) 21.3  (86) 42  (21) 7.5  (82)                         3347W92 120 19.4  (65) 0.8  (59) 14.2  (54) 207  (52) 0.8  (59)                                                               7 30 12.1 (100) 6.9                                                           (100) 127.4 (100)   6.9                                                        (100)                            3855W92 120 91.8 (100) 1.5 (100) 24.1  (97)   1.5 (100)                        8 30 5.2  (93) 6.8  (75) 364.5  (77) 150.2  (71)                               250W93 120 27.3  (57) 235.1  (56) 31.7  (35) 646.2  (55)                       9 30 2.6 (100) 4.1 (100) 377.9  (97) 1104.1  (97)                              637W93 120 6.2 (100) 108.3  (99) 211.0  (93) 1505.7  (94)                      (BALB/C) 30     420.3  (98) 1604.27  (98)                                      10 30 19.3  (65.35) 3.0  (65) 17.3  (16)   3.0  (65)                           1311W92 120 1.1  (21.6) ND  ND  (0)   ND                                       11 30 2.7  (74.45) 4.5 (100) 301.3  (86.725) 426.8  (92.275) 4.8 (100)                                                        3352W93 120 4.4  (84.95)                                                      0.8 (100) 140.0  (67.7)                                                        389.8  (67.925) 2.4                                                            (88.1)                           12 30 ND  16.3 (100) 550.8  (91) 1205  (93) 1.6  (92.8)                        1834W93 120 14.7  (66.925) 402.3  (80.7) 44.9  (67) 1941  (82) ND                                                             13 30 7.9 (100) 14.4                                                          (100) 373.7 (100) 665.8                                                        (100)                            5755U93 120 12.5 (100) 4.8 (100) 322.1 (100) 738.4 (100)                       14 30 9.4 (100) 4.1 (100) 218.3 (100) 535.0  (90.675)                          4204W93 120 34.6  (76.775) 65.3  (48.75) 127.8 (100) 932.8  (88.05)          __________________________________________________________________________      Prodrugs 1 to 14 shown in TABLE 1 are as follows:                              1.                                                                             N(N-(4(((1,2-dihydro-3-methyl-1-oxobenzo(F)quinazolin-9-yl)methyl)amino)-     -fluorobenzoyl)-L-glutam-1-yl)-L-glutamic acid                                  2.                                                                             N((S)-4-carboxy-2-(5-(((1,2-dihydro-3-methyl-1-oxoxbenzo(F)quinazolin-9-y     )methyl)amino)-1-oxo-2-isoindolinyl)butanoyl)-L-phenylalanine                   3.                                                                             N(N-(2-fluoro-4-(((1,2-dihydro-3-methyl-1-oxobenzo(F)quinazolin-9-yl)meth     l)amino)benzoyl)-L-glutam-1-yl)-3-(1-naphthyl)-L-alanine                        4.                                                                             N(N-(4(((1,2-dihydro-3-methyl-1-oxobenzo(F)quinazolin-9-yl)methyl)amino)-     -fluorobenzoyl)-L-glutam-1-yl)-2-carboxy-L-phenylalanine                        5.                                                                             N(N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(F)quinazolin-9-yl)methyl)amino)     2-fluorobenzoyl)-L-glutam-1-yl-2-iodo-L-phenylalanine                           6.                                                                             N(4-((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-     -aspartic acid                                                                  7.                                                                             N(4-((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-     -carboxy-L-phenylalanine                                                        8.                                                                             N(4-((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-     -cyclopentyl-L-phenylalanine                                                    9.                                                                             N(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl     3-cyclopentyl-L-phenylalanine                                                   10.                                                                            N(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl     L-phenylalanine.                                                                11.                                                                            N(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl     3-cyclobutyl-L-phenylalanine.                                                   12.                                                                            N(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl     3-tert-butyl-L-phenylalanine.                                                   13.                                                                            N(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl     3-cyclopentyl-L-tyrosine.                                                       14.                                                                            N(4-((3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl     -L-glutam-1-yl-3-cyclopentyl-L-phenylalanine                              

EXAMPLE 219 Molecular Modelling of Human CPA1

The Brookhaven Protein Database (Bernstein F. C. et al., J. Mol. Biol. 112, 535-542 [1977]) was searched and bovine CPA selected as that likely to have the greatest structural similarity to human CPA (HCPA) based on the base sequence homology. The COMPOSER algorithm which was used to develop the HCPA model is part of the commercially available SYBYL molecular modelling package sold by TRIPOS Associates (SYBYL Molecular Modeling Software, Version 5.3, November 1989, TRIPOS Associates Inc., St. Louis, Mo. 63144). The COMPOSER algorithm was used to fit the amino acid residues of the human CPA sequence into the 3D coordinates of the crystal structure of bovine CPA to determine a best fit and consequently a projected 3D structure for HCPA.

EXAMPLE 220 Cell Culture Targetting and Therapy

Wein-133 B-cell lymphoma cells were incubated with a conjugate of wild-type HCPA1 and Campath for 20 min. Cells were then washed to remove unbound conjugate and returned to cell culture at 500,000 cells ml⁻¹. Cells were cultured in the presence of varying concentrations of the prodrug MTX-Phe and levels of inhibition of growth analysed after 3 days. Results are illustrated in FIG. 1. Similar results were obtained when a conjugate of mutant 268 Gly with Campath was substituted for the wild type conjugate.

EXAMPLE 221 Prodrug Toxicity

The stable prodrug of the present invention should in addition to being stable in vivo also be less toxic in the host than the parent drug in order to permit larger dosing levels of prodrug and in turn generation of local high concentrations of active agent. We have found that the maximum tolerated dose for methotrexate in the mouse (female, strain CD-1 nu/nu) is 4 mg/kg IV once daily for 5 days. Doses of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-phenylalanine up to 20 mg/kg IV once daily for 5 days were tolerated, and doses of up to 40 mg/kg of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine or N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tert-butyl-L-phenylalanine IV once daily for 5 days produced no toxicity; therefore the maximally tolerated doses of the latter two compounds are greater than 40 mg/kg. In Swiss nu/nu mice, the maximum tolerated dose of methotrexate is 2.5 mg/kg IV daily for 5 days. In these mice, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-tyrosine was tolerated at doses to 50 mg/kg IV daily for 5 days while N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclobutyl-L-phenylalanine showed some toxicity at does of 30 mg/kg IV daily for 5 days. Thus, in Swiss nu/nu mice, with this daily ×5 schedule, the MTD for N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-tyrosine and N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclobutyl-L-phenylalanine would be >50 mg/kg and <30 mg/kg, respectively. Thus the prodrugs of the present invention are indeed much better tolerated than the corresponding active agent.

EXAMPLE 222 Activity of a Mutant Human Carboxypeptidase A1 with a Stable Prodrug

As indicated herein, the in vivo stable prodrug of the present invention should be a better substrate for a mutant enzyme than the corresponding wild type enzyme in order for the stable prodrug to be activated selectively at the tumour. Table 2 shows the enzyme kinetics for several prodrugs of methotrexate with wild type human carboxypeptidase A1 and mutants with thr 268 mutated to either Ala or Gly. The most relevant kinetic constant in the table is the kcat/Km, which provides an estimate of the second order rate constant for the enzyme catalyzed conversion of prodrug to methotrexate. Thus, the larger the kcat/Km, the more efficient the enzyme with particular substrate. It can be seen that the kcat/Km for human CPA1 with one of the best substrates for this wild type enzyme N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-phenylalanine the prodrug of MTX, is 0.44 /uM/sec. The kcat/Km values for N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-2-cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tertbutyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclobutyl-L-phenylalanine, and N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl-L-glutam-1-yl-3-cyclopentyl-L-tyrosine for the human CPA1 are much lower; indeed the human CPA catalyzed reactions with the latter four prodrugs were not measureable. However, the kcat/Km for these five stable prodrugs with 268 Gly are 0.157, 0.092, 0.38, 1.8 and 0.18/uM/sec, respectively. Thus, the single mutations at 268 from thr to gly converted very poor substrates in the wild type enzyme to excellent substrates in the mutant enzyme. Indeed in the case of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-tertbutyl-L-phenylalanine the activity of 268 Gly with the prodrug is not different than the human CPA1 with N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-phenylalanine, one of its best substrates. Further, in the case of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-3-cyclobutyl-L-phenylalanine the activity of 268 Gly is 4 times better than CPA1 with N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-1-yl-L-phenylaline. Thus, the compounds are much better substrates for the mutant enzyme than the wild type enzyme which permits very specific tumor targeted activation of the nontoxic, stable prodrug to the active agent.

                                      TABLE 2                                      __________________________________________________________________________                       kinetic                                                                              Human                                                    Prodrug constant CPA1 268ala 268gly                                          __________________________________________________________________________     N-(4-(((2,4-diamino-6-pteridinyl)methyl)                                                         Km (uM)                                                                              4.3  0.9  0.3                                            methylamino)benzoyl)-L-glutam-1-yl- kcat (1/sec) 1.9 2.0 2.2                   L-phenylalanine kcat/Km 0.44 2.25 7.35                                         N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) 61 56 15                      methylamino)benzoyl)-L-glutam-1-yl-2- kcat (1/sec) 0.012 0.088 2.35                                             cyclopentyl-L-phenylalanine kcat/Km                                           0.0002 0.0016 0.157                            N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) ND 31 15                      methylamino)benzoyl)-L-glutam-1-yl-3- kcat (1/sec) ND 0.001 1.4                                                 cyclopentyl-L-phenylalanine kcat/Km ND                                        0.00003 0.092                                  N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) ND ND 2.1                     methylamino)benzoyl)-L-glutam-1-yl-3- kcat (1/sec) ND ND 0.81                  tertbutyl-L-phenylalanine kcat/K/m ND ND 0.38                                  N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) ND 160 1.8                    methylamino)benzoyl)-L-glutam-1-yl-3- kcat (1/sec) ND 0.12 3.0                 cyclobutyl-L-phenylalanine kcat/Km ND 0.00075 1.8                              N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) ND 50 18                      methylamino)benzoyl)-L-glutam-1-yl-3 kcat (1/sec) ND 0.0006 3.0                                                 cyclopentyl-L-tyrosine kcat/Km ND                                             0.00001 0.18                                 __________________________________________________________________________      ND = not determined; rate to slow to assay. kcat ≦ 0.0005/sec.    

Kinetics were determined by a modification of the coupled assay of Kuefner, et al (1989) Biochemistry 28 2288-2297 in which the product MTX is converted by excess carboxypeptidase G to the pteroate of MTX. In this assay the reaction is followed spectrophotometrically at 315 nm; at this wavelength the change in molar absorbtivity for the coupled reaction was determined to be 9.57 mM⁻¹ cm⁻¹. Assays were performed at 25° C. in 25 mM tris-HCl, 100 mM NaCl, pH 7.4. Prodrugs were preincubated at 25° C. with buffer and 17 milliunits of Sigma #C-9658 carboxypeptidase G in 1 ml total volume. Assay was initiated with carboxypeptidase A or the appropriate mutant. Initial velocities were obtained, and standard Michaelis-Menten calculations were used to determine kinetic constants (in Biochemistry, Lehninger, A. L. 1970, Worth Publishers, Inc, NY).

                                      TABLE 3                                      __________________________________________________________________________     Antibodies and Antigens                                                        Antigen      Antibody     Current uses                                         __________________________________________________________________________     Tumor associated                                                                 Antigens                                                                       Pan Adenocarcinoma ING-1 Imaging and therapy of                                 (Wellcome) various carcinomas.                                                Cytokeratins c174 Imaging and therapy of                                        (Biomira) Squamous cell carcinomas.                                           Cytokeratins 16.88 Imaging and therapy of                                       (Organon Teknika) Squamous cell carcinomas.                                   Carcinoembryonic Antigen NR-CO-02 Imaging and therapy of                        (NeoRex) colon/gastrointestinal                                                 tumors                                                                       Carcinoembryonic Antigen PR.1A3 Imaging and therapy of                          (ICRF) colon/gastrointestinal                                                   tumors                                                                       Carcinoembryonic Antigen Immu-14 Imaging and therapy of                         (Immunomedics) colon/gastrointestinal                                           tumors                                                                       Carcinoembryonic Antigen Col-1 Imaging and therapy of                           (Dow) colon/gastrointestinal                                                    tumors                                                                       Carcinoembryonic Antigen C110 Imaging and therapy of                            (Abbott) colon/gastrointestinal                                                 tumors                                                                       Carcinoembryonic Antigen A5B7 Imaging and therapy of                            (Brit. J. Cancer, 1986, colon/gastrointestinal                                 54, 75) tumors                                                                Carcinoembryonic Antigen BW431/26 Imaging and therapy of                        (Br J. Cancer 1992, 65, 234) colon/gastrointestinal                            (Cancer Immunol tumors                                                         Immunother 1992, 34, 343)                                                     Carcinoembryonic Antigen C46.3 Imaging and therapy of                           (Cytogen) colon/gastrointestinal                                                tumors                                                                       Pan Adenocarcinoma MAb 12.8 Imaging and therapy of                              (Biochem Pharmacol 1991, various carcinomas.                                   42, 2062)                                                                     Pan Adenocarcinoma CC49 and B72.3 Imaging and therapy of                       (TAG-72) (Dow) various carcinomas.                                             Histones TNT-1 and TNT-2 All tumors with necrosis                               (Techniclone)                                                                 Folate binding Protein MOV18 Ovarian Carcinoma                                  (Centocor)                                                                    Folate binding Protein MOV19 Ovarian Carcinoma                                  (Int. J. Cancer 39, 297,                                                       1987)                                                                         Pan Adenocarcinoma 323/A3 Imaging and therapy of                                (Centocor) various carcinomas.                                                Pan Adenocarcinoma 17.1A Imaging and therapy of                                 (Centocor) various carcinomas.                                                Renal cell G250 Imaging and therapy of                                          (Centocor) Renal Cell carcinomas.                                             200 Kd squamous cell U36 Imaging and therapy of                                surface antigen (Centocor) Squamous cell carcinomas.                           22 Kd squamous cell surface E48 Imaging and therapy of                         antigen (Centocor) Squamous cell carcinomas.                                   125 Kd colon SF-25 Imaging and therapy of                                      adenocarcinoma cell surface (Centocor) various carcinomas.                     glycoprotein                                                                   High molecular weight Various mucin targeting Imaging and therapy of                                    mucins antibodies (Trends various carcinomas.                                   Biochemical Science, 1992,                            17, 359)                                                                      High molecular weight HMFG1 Imaging and therapy of                             Mucin (Cancer Research, 1992, 52, various carcinomas.                           904)                                                                          High molecular weight BrE-3 Imaging and therapy of                             Mucin (Hybridoma, 1993, 12, 15) various carcinomas.                            Lung squamous cell RS7-3G11 Imaging and therapy of lung                        carcinoma antigen (Antibody, squamous cell carcinomas.                          Immunoconjugates and                                                           Radiopharmaceuticals, 1991                                                     4 703)                                                                        LE.sup.Y BR64 and BR96 Imaging and therapy of                                  related tumor antigen (Bristol-Meyers Squibb) various carcinomas.                                       HER2 protooncogene P185HER2 Imaging and                                       therapy of                                             product (NeoRx) various carcinomas,                                              especially breast,                                                           human carcinoma antigen 15A82a Imaging and therapy of                           (Cytogen) various carcinomas.                                                 human Chorionic W14A and SB10 Imaging and therapy of                           gonadotropin (Int J. Cancer, 1984, 33, 429 various carcinomas.                  Br. J. Cancer, 1990, 61, 659)                                               __________________________________________________________________________

                                      TABLE 4                                      __________________________________________________________________________     Amino acid coding sequences for human CPA1 and CPA2.*                          __________________________________________________________________________                -110  -100  -90   -80   -70                                           HCPA1MRGLLVLSVLLGAVFGKEDFVGHQVLRISVADEAQVQKVKELEDLEHLQL                        HCPA2::LI:FFGA:F:HIYCL:T:::D:::E:VPSN:E:IKNLLQ::AQ:::::                         - -60-50-40-30-°                                                       HCPA1DFWRGPAHPGSPIDVRMPFPSIQAVKIFLESHGISYETMIEDVQSLLDEE                        HCPA2:::KS:TTS:ETAH::V::VNV::::V::::Q::A:SI::::::V:::K:                         - -101112131                                                                  HCPA1QEQMFAFRSRARSTDTFNYATYHTLEEIYDFLDLLVAENPHLVSKIQIGN                        HCPA2N:E:LFN:R:E:-SGN::FGA:::::::SQEM:N::::H:G::::VN::S                         - 4151617181                                                                  HCPA1TYEGRPIYVLKFSTGGSKRPAIWIDTGIHSREWVTQASGVWFAKKITQDY                        HCPA2SF:N::MN::::::::-DK::::L:A:::A:::::::TAL:T:N::VS::                         - 91101111121131                                                              HCPA1GQDAAFTAILDTLDIFLEIVTNPDGFAFTHSTNRMWRKTRSHTAGSLCIG                        HCPA2:K:PSI:S:::A:::::LP::::::YV:SQTK:::::::::KVS::::V:                         - 141151161171181                                                             HCPA1VDPNRNWDAGFGLSGASSNPCSETYHGKFANSEVEVKSIVDFVKDHGNIK                        HCPA2::::::::::::GP:A::::::DS:::PS:::::::::::::I:S::KV:                         - 191201211221231                                                             HCPA1AFISIHSYSQLLMYPYGYKTEPVPDQDELDQLSKAAVTALASLYGTKFNY                        HCPA2:::TL::::::::F:::::CTKLD:F:::SEVAQK:AQS:R::H:::YKV                         - 241251261271281                                                             HCPA1GSIIKAIYQASGSTIDWTYSQGIKYSFTFELRDTGRYGFLLPASQIIPTA                        HCPA2:P:CSV::::::GS:::S:DY::::::A:::::::::::::::R::L:::                         - 291301                                                                      HCPA1(SEQ ID NO:2)KETWLALLTIMEHTLNHRY                                          HCPA2(SEQ ID NO:4)E::::F:KA::::VRD:::                                        __________________________________________________________________________      *Numbering scheme for CPA1 is used. Dashes at positions 3 and 57 indicate      a gap in the  A2 sequence required for optimal alignment with A1.              Colons indicate identity between A1 and A2.                                    PreproCPA begins at amino acid -110 and runs to +309.                          ProCPA begins at amino acid -94 and runs to +309.                              mature CPA begins at amino acid +1 and runs to +309.                     

                                      TABLE 5                                      __________________________________________________________________________     Amino acid sequences for the 268Gly mutations of human CPA1 and CPA2.*         __________________________________________________________________________     (mut.)       -110  -100  -90   -80   -70                                         HCPA1MRGLLVLSVLLGAVFGKEDFVGHQVLRISVADEAQVQKVKELEDLEHLQL                        HCPA2::LI:FFGA:F:HIYCL:T:::D:::E:VPSN:E:IKNLLQ::AQ:::::                         - (mut.)-60-50-40-30-20                                                       HCPA1DFWRGPAHFGSPIDVRMPFPSIQAVKIFLESHGISYETMIEDVQSLLDEE                        HCPA2:::KS:TTS:ETAH::V::VNV::::V::::Q::A:SI::::::V:::K:                         - (mut.)-101112131                                                            HCPA1QEQMFAFRSRARSTDTFNYATYHTLEEIYDFLDLLVAENPHLVSKIQIGN                        HCPS2N:E:LFN:R:E:-SGN::FGA:::::::SQEM:N::::H:G::::VN::S                         - (mut.)4151617181                                                            HCPA1TYEGRPIYVLKFSTGGSKRPAIWIDTGIHSREWVTQASGVWFAKKITQDY                        HCPA2SF:N::MN::::::::-DK::::L:A:::A:::::::TAL:T:N::VS::                         - (mut.)91101111121131                                                        HCPA1GQDAAFTAILDTLDIFLEIVTNPDGFAFTHSTNRMWRKTRSHTAGSLCIG                        HCPA2:K:PSI:S:::A:::::LP::::::YV:SQTK:::::::::KVS::::V:                         - (mut.)141151161171181                                                       HCPA1VDPNRNWDAGFGLSGASSNPCSETYHGKFANSEVEVKSIVDFVKDHGNIK                        HCPA2::::::::::::GP:A::::::DS:::PS:::::::::::::I:S::KV:                         - (mut.)191201211221231                                                       HCPA1AFISIHSYSQLLMYPYGYKTEPVPDQDELDQLSKAAVTALASLYGTKFNY                        HCPA2:::TL::::::::F:::::CTKLD:F:::SEVAQK:AQS:R::H:::YKV                         - (mut.)241251261271281                                                       HCPA1GSIIKAIYQASGSTIDWTYSQGIKYSFGFELRDTGRYGFLLPASQIIPTA                        HCPA2:P:CSV::::::GS:::S:DY::::::G:::::::::::::::R::L:::                         - (mut.)291301                                                                HCPA1(SEQ ID NO:17)KETWLALLTIMEHTLNHPY                                         HCPA2(SEQ ID NO:19)E::::G:KA::::VRD:::                                       __________________________________________________________________________      *Numbering scheme for CPA1 is used. Dashes at positions 3 and 57 indicate      a gap in the A2 sequence required for optimal alignment with A1.               Colons indicate identity between A1 and A2.                              

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - <160> NUMBER OF SEQ ID NOS: 22                                        - - <210> SEQ ID NO 1                                                         <211> LENGTH: 1257                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Homo sapiens                                                   <220> FEATURE:                                                                 <221> NAME/KEY: CDS                                                            <222> LOCATION: (1)..(1257)                                                     - - <400> SEQUENCE: 1                                                          - - atg cgc ggg ttg ctg gtg ttg agt gtc ctg tt - #g ggg gct gtc ttt         ggc       48                                                                     Met Arg Gly Leu Leu Val Leu Ser Val Leu Le - #u Gly Ala Val Phe Gly             1               5 - #                 10 - #                 15               - - aag gag gac ttt gtg ggg cat cag gtg ctc cg - #a atc tct gta gcc gat            96                                                                        Lys Glu Asp Phe Val Gly His Gln Val Leu Ar - #g Ile Ser Val Ala Asp                         20     - #             25     - #             30                   - - gag gcc cag gta cag aag gtg aag gag ctg ga - #g gac ctg gag cac ctg           144                                                                        Glu Ala Gln Val Gln Lys Val Lys Glu Leu Gl - #u Asp Leu Glu His Leu                     35         - #         40         - #         45                       - - cag ctg gac ttc tgg cgg ggc cct gcc cac cc - #t ggc tcc ccc atc gac           192                                                                        Gln Leu Asp Phe Trp Arg Gly Pro Ala His Pr - #o Gly Ser Pro Ile Asp                 50             - #     55             - #     60                           - - gtc cga atg ccc ttc ccc agc atc cag gcg gt - #c aag atc ttt ctg gag           240                                                                        Val Arg Met Pro Phe Pro Ser Ile Gln Ala Va - #l Lys Ile Phe Leu Glu             65                 - # 70                 - # 75                 - # 80        - - tcc cac ggc atc agc tat gag acc atg atc ga - #g gac gtg cag tcg ctg           288                                                                        Ser His Gly Ile Ser Tyr Glu Thr Met Ile Gl - #u Asp Val Gln Ser Leu                             85 - #                 90 - #                 95               - - ctg gac gag gag cag gag cag atg ttc gcc tt - #c cgg tcc cgg gcg cgc           336                                                                        Leu Asp Glu Glu Gln Glu Gln Met Phe Ala Ph - #e Arg Ser Arg Ala Arg                        100      - #           105      - #           110                   - - tcc acc gac act ttt aac tac gcc acc tac ca - #c acc ctg gag gag atc           384                                                                        Ser Thr Asp Thr Phe Asn Tyr Ala Thr Tyr Hi - #s Thr Leu Glu Glu Ile                    115          - #       120          - #       125                       - - tat gac ttc ctg gac ctg ctg gtg gcg gag aa - #c ccg cac ctt gtc agc           432                                                                        Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu As - #n Pro His Leu Val Ser                130              - #   135              - #   140                           - - aag atc cag att ggc aac acc tat gaa ggg cg - #t ccc att tac gtg ctg           480                                                                        Lys Ile Gln Ile Gly Asn Thr Tyr Glu Gly Ar - #g Pro Ile Tyr Val Leu            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - aag ttc agc acg ggg ggc agt aag cgt cca gc - #c atc tgg atc gac         acg      528                                                                     Lys Phe Ser Thr Gly Gly Ser Lys Arg Pro Al - #a Ile Trp Ile Asp Thr                           165  - #               170  - #               175               - - ggc atc cat tcc cgg gag tgg gtc acc cag gc - #c agt ggg gtc tgg ttt           576                                                                        Gly Ile His Ser Arg Glu Trp Val Thr Gln Al - #a Ser Gly Val Trp Phe                        180      - #           185      - #           190                   - - gca aag aag atc act caa gac tat ggg cag ga - #t gca gct ttc acc gcc           624                                                                        Ala Lys Lys Ile Thr Gln Asp Tyr Gly Gln As - #p Ala Ala Phe Thr Ala                    195          - #       200          - #       205                       - - att ctc gac acc ttg gac atc ttc ctg gag at - #c gtc acc aac cct gat           672                                                                        Ile Leu Asp Thr Leu Asp Ile Phe Leu Glu Il - #e Val Thr Asn Pro Asp                210              - #   215              - #   220                           - - ggc ttt gcc ttc acg cac agc acg aat cgc at - #g tgg cgc aag act cgg           720                                                                        Gly Phe Ala Phe Thr His Ser Thr Asn Arg Me - #t Trp Arg Lys Thr Arg            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - tcc cac aca gca ggc tcc ctc tgt att ggc gt - #g gac ccc aac agg         aac      768                                                                     Ser His Thr Ala Gly Ser Leu Cys Ile Gly Va - #l Asp Pro Asn Arg Asn                           245  - #               250  - #               255               - - tgg gac gct ggc ttt ggg ttg tcc gga gcc ag - #c agt aac ccc tgc tcg           816                                                                        Trp Asp Ala Gly Phe Gly Leu Ser Gly Ala Se - #r Ser Asn Pro Cys Ser                        260      - #           265      - #           270                   - - gag act tac cac ggc aag ttt gcc aat tcc ga - #a gtg gag gtc aag tcc           864                                                                        Glu Thr Tyr His Gly Lys Phe Ala Asn Ser Gl - #u Val Glu Val Lys Ser                    275          - #       280          - #       285                       - - att gta gac ttt gtg aag gac cat ggg aac at - #c aag gcc ttc atc tcc           912                                                                        Ile Val Asp Phe Val Lys Asp His Gly Asn Il - #e Lys Ala Phe Ile Ser                290              - #   295              - #   300                           - - atc cac agc tac tcc cag ctc ctc atg tat cc - #c tat ggc tac aaa aca           960                                                                        Ile His Ser Tyr Ser Gln Leu Leu Met Tyr Pr - #o Tyr Gly Tyr Lys Thr            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - gaa cca gtc cct gac cag gat gag ctg gat ca - #g ctt tcc aag gct         gct     1008                                                                     Glu Pro Val Pro Asp Gln Asp Glu Leu Asp Gl - #n Leu Ser Lys Ala Ala                           325  - #               330  - #               335               - - gtg aca gcc ctg gcc tct ctc tac ggg acc aa - #g ttc aac tat ggc agc          1056                                                                        Val Thr Ala Leu Ala Ser Leu Tyr Gly Thr Ly - #s Phe Asn Tyr Gly Ser                        340      - #           345      - #           350                   - - atc atc aag gca att tat caa gcc agt gga ag - #c act att gac tgg acc          1104                                                                        Ile Ile Lys Ala Ile Tyr Gln Ala Ser Gly Se - #r Thr Ile Asp Trp Thr                    355          - #       360          - #       365                       - - tac agc cag ggc atc aag tac tcc ttc acc tt - #c gag ctc cgg gac act          1152                                                                        Tyr Ser Gln Gly Ile Lys Tyr Ser Phe Thr Ph - #e Glu Leu Arg Asp Thr                370              - #   375              - #   380                           - - ggg cgc tat ggc ttc ctg ctg cca gcc tcc ca - #g atc atc ccc aca gcc          1200                                                                        Gly Arg Tyr Gly Phe Leu Leu Pro Ala Ser Gl - #n Ile Ile Pro Thr Ala            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - aag gag acg tgg ctg gcg ctt ctg acc atc at - #g gag cac acc ctg         aat     1248                                                                     Lys Glu Thr Trp Leu Ala Leu Leu Thr Ile Me - #t Glu His Thr Leu Asn                           405  - #               410  - #               415               - - cac ccc tac              - #                  - #                        - #       1257                                                                   His Pro Tyr                                                                     - -  - - <210> SEQ ID NO 2                                                    <211> LENGTH: 419                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Homo sapiens                                                    - - <400> SEQUENCE: 2                                                          - - Met Arg Gly Leu Leu Val Leu Ser Val Leu Le - #u Gly Ala Val Phe         Gly                                                                                1               5 - #                 10 - #                 15              - - Lys Glu Asp Phe Val Gly His Gln Val Leu Ar - #g Ile Ser Val Ala Asp                    20     - #             25     - #             30                   - - Glu Ala Gln Val Gln Lys Val Lys Glu Leu Gl - #u Asp Leu Glu His Leu                35         - #         40         - #         45                       - - Gln Leu Asp Phe Trp Arg Gly Pro Ala His Pr - #o Gly Ser Pro Ile Asp            50             - #     55             - #     60                           - - Val Arg Met Pro Phe Pro Ser Ile Gln Ala Va - #l Lys Ile Phe Leu Glu        65                 - # 70                 - # 75                 - # 80        - - Ser His Gly Ile Ser Tyr Glu Thr Met Ile Gl - #u Asp Val Gln Ser Leu                        85 - #                 90 - #                 95               - - Leu Asp Glu Glu Gln Glu Gln Met Phe Ala Ph - #e Arg Ser Arg Ala Arg                   100      - #           105      - #           110                   - - Ser Thr Asp Thr Phe Asn Tyr Ala Thr Tyr Hi - #s Thr Leu Glu Glu Ile               115          - #       120          - #       125                       - - Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu As - #n Pro His Leu Val Ser           130              - #   135              - #   140                           - - Lys Ile Gln Ile Gly Asn Thr Tyr Glu Gly Ar - #g Pro Ile Tyr Val Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Lys Phe Ser Thr Gly Gly Ser Lys Arg Pro Al - #a Ile Trp Ile Asp         Thr                                                                                              165  - #               170  - #               175              - - Gly Ile His Ser Arg Glu Trp Val Thr Gln Al - #a Ser Gly Val Trp Phe                   180      - #           185      - #           190                   - - Ala Lys Lys Ile Thr Gln Asp Tyr Gly Gln As - #p Ala Ala Phe Thr Ala               195          - #       200          - #       205                       - - Ile Leu Asp Thr Leu Asp Ile Phe Leu Glu Il - #e Val Thr Asn Pro Asp           210              - #   215              - #   220                           - - Gly Phe Ala Phe Thr His Ser Thr Asn Arg Me - #t Trp Arg Lys Thr Arg       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ser His Thr Ala Gly Ser Leu Cys Ile Gly Va - #l Asp Pro Asn Arg         Asn                                                                                              245  - #               250  - #               255              - - Trp Asp Ala Gly Phe Gly Leu Ser Gly Ala Se - #r Ser Asn Pro Cys Ser                   260      - #           265      - #           270                   - - Glu Thr Tyr His Gly Lys Phe Ala Asn Ser Gl - #u Val Glu Val Lys Ser               275          - #       280          - #       285                       - - Ile Val Asp Phe Val Lys Asp His Gly Asn Il - #e Lys Ala Phe Ile Ser           290              - #   295              - #   300                           - - Ile His Ser Tyr Ser Gln Leu Leu Met Tyr Pr - #o Tyr Gly Tyr Lys Thr       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Glu Pro Val Pro Asp Gln Asp Glu Leu Asp Gl - #n Leu Ser Lys Ala         Ala                                                                                              325  - #               330  - #               335              - - Val Thr Ala Leu Ala Ser Leu Tyr Gly Thr Ly - #s Phe Asn Tyr Gly Ser                   340      - #           345      - #           350                   - - Ile Ile Lys Ala Ile Tyr Gln Ala Ser Gly Se - #r Thr Ile Asp Trp Thr               355          - #       360          - #       365                       - - Tyr Ser Gln Gly Ile Lys Tyr Ser Phe Thr Ph - #e Glu Leu Arg Asp Thr           370              - #   375              - #   380                           - - Gly Arg Tyr Gly Phe Leu Leu Pro Ala Ser Gl - #n Ile Ile Pro Thr Ala       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Lys Glu Thr Trp Leu Ala Leu Leu Thr Ile Me - #t Glu His Thr Leu         Asn                                                                                              405  - #               410  - #               415              - - His Pro Tyr                                                                - -  - - <210> SEQ ID NO 3                                                    <211> LENGTH: 1251                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Homo sapiens                                                   <220> FEATURE:                                                                 <221> NAME/KEY: CDS                                                            <222> LOCATION: (1)..(1251)                                                     - - <400> SEQUENCE: 3                                                          - - atg agg ttg atc ctg ttt ttt ggt gcc ctt tt - #t ggg cat atc tac tgt            48                                                                        Met Arg Leu Ile Leu Phe Phe Gly Ala Leu Ph - #e Gly His Ile Tyr Cys              1               5 - #                 10 - #                 15               - - cta gaa aca ttt gtg gga gac caa gtt ctt ga - #g att gta cca agc aat            96                                                                        Leu Glu Thr Phe Val Gly Asp Gln Val Leu Gl - #u Ile Val Pro Ser Asn                         20     - #             25     - #             30                   - - gaa gaa caa att aaa aat ctg cta caa ttg ga - #g gct caa gaa cat ctc           144                                                                        Glu Glu Gln Ile Lys Asn Leu Leu Gln Leu Gl - #u Ala Gln Glu His Leu                     35         - #         40         - #         45                       - - cag ctt gat ttt tgg aaa tca ccc acc acc tc - #a ggg gag aca gcc cac           192                                                                        Gln Leu Asp Phe Trp Lys Ser Pro Thr Thr Se - #r Gly Glu Thr Ala His                 50             - #     55             - #     60                           - - gtc cga gtt ccc ttc gtc aac gtc cag gca gt - #c aaa gtg ttc ttg gag           240                                                                        Val Arg Val Pro Phe Val Asn Val Gln Ala Va - #l Lys Val Phe Leu Glu             65                 - # 70                 - # 75                 - # 80        - - tcc cag gga att gcc tat tcc atc atg att ga - #a gac gtg cag gtc ctg           288                                                                        Ser Gln Gly Ile Ala Tyr Ser Ile Met Ile Gl - #u Asp Val Gln Val Leu                             85 - #                 90 - #                 95               - - ttg gac aaa gag aat gaa gaa atg ctt ttt aa - #t agg aga aga gaa cgg           336                                                                        Leu Asp Lys Glu Asn Glu Glu Met Leu Phe As - #n Arg Arg Arg Glu Arg                        100      - #           105      - #           110                   - - agt ggt aac ttc aat ttt ggg gcc tac cat ac - #c ctg gaa gag att tcc           384                                                                        Ser Gly Asn Phe Asn Phe Gly Ala Tyr His Th - #r Leu Glu Glu Ile Ser                    115          - #       120          - #       125                       - - caa gaa atg gat aac ctc gtg gct gag cac cc - #t ggt cta gtg agc aaa           432                                                                        Gln Glu Met Asp Asn Leu Val Ala Glu His Pr - #o Gly Leu Val Ser Lys                130              - #   135              - #   140                           - - gtg aat att ggc tct tct ttt gag aac cgg cc - #t atg aac gtg ctc aag           480                                                                        Val Asn Ile Gly Ser Ser Phe Glu Asn Arg Pr - #o Met Asn Val Leu Lys            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - ttc agc acc gga gga gac aag cca gct atc tg - #g ctg gat gcg ggg         atc      528                                                                     Phe Ser Thr Gly Gly Asp Lys Pro Ala Ile Tr - #p Leu Asp Ala Gly Ile                           165  - #               170  - #               175               - - cat gct cga gag tgg gtt aca caa gct acg gc - #a ctt tgg aca gca aat           576                                                                        His Ala Arg Glu Trp Val Thr Gln Ala Thr Al - #a Leu Trp Thr Ala Asn                        180      - #           185      - #           190                   - - aag att gtt tct gat tat gga aag gac cca tc - #c atc act tcc att ctg           624                                                                        Lys Ile Val Ser Asp Tyr Gly Lys Asp Pro Se - #r Ile Thr Ser Ile Leu                    195          - #       200          - #       205                       - - gac gcc ctg gat atc ttc ctc ctg cca gtc ac - #a aac cct gat gga tac           672                                                                        Asp Ala Leu Asp Ile Phe Leu Leu Pro Val Th - #r Asn Pro Asp Gly Tyr                210              - #   215              - #   220                           - - gtg ttc tct caa acc aaa aat cgt atg tgg cg - #g aag acc cgg tcc aag           720                                                                        Val Phe Ser Gln Thr Lys Asn Arg Met Trp Ar - #g Lys Thr Arg Ser Lys            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - gta tct gga agc ctc tgt gtt ggt gtg gat cc - #t aac cgg aac tgg         gat      768                                                                     Val Ser Gly Ser Leu Cys Val Gly Val Asp Pr - #o Asn Arg Asn Trp Asp                           245  - #               250  - #               255               - - gca ggt ttt gga gga cct gga gcc agc agc aa - #c cct tgc tct gat tca           816                                                                        Ala Gly Phe Gly Gly Pro Gly Ala Ser Ser As - #n Pro Cys Ser Asp Ser                        260      - #           265      - #           270                   - - tac cac gga ccc agt gcc aac tct gaa gtt ga - #a gtg aaa tcc ata gtg           864                                                                        Tyr His Gly Pro Ser Ala Asn Ser Glu Val Gl - #u Val Lys Ser Ile Val                    275          - #       280          - #       285                       - - gac ttc atc aag agt cat gga aaa gtc aag gc - #c ttc att acc ctc cac           912                                                                        Asp Phe Ile Lys Ser His Gly Lys Val Lys Al - #a Phe Ile Thr Leu His                290              - #   295              - #   300                           - - agc tat tcc cag ctg ctg atg ttc ccc tat gg - #g tac aaa tgt acc aag           960                                                                        Ser Tyr Ser Gln Leu Leu Met Phe Pro Tyr Gl - #y Tyr Lys Cys Thr Lys            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - tta gat gac ttt gat gag ctg agt gaa gtg gc - #c caa aag gct gcc         caa     1008                                                                     Leu Asp Asp Phe Asp Glu Leu Ser Glu Val Al - #a Gln Lys Ala Ala Gln                           325  - #               330  - #               335               - - tct ctg aga agc ctg cat ggc acc aag tac aa - #a gtg gga cca atc tgc          1056                                                                        Ser Leu Arg Ser Leu His Gly Thr Lys Tyr Ly - #s Val Gly Pro Ile Cys                        340      - #           345      - #           350                   - - tct gtc atc tac caa gcc agt gga gga agc at - #t gac tgg tcc tat gat          1104                                                                        Ser Val Ile Tyr Gln Ala Ser Gly Gly Ser Il - #e Asp Trp Ser Tyr Asp                    355          - #       360          - #       365                       - - tat ggc atc aag tac tca ttt gcc ttt gaa ct - #g aga gac aca ggg cgc          1152                                                                        Tyr Gly Ile Lys Tyr Ser Phe Ala Phe Glu Le - #u Arg Asp Thr Gly Arg                370              - #   375              - #   380                           - - tac ggc ttc ctc ttg cca gcc cgt cag atc ct - #g ccc aca gcc gag gag          1200                                                                        Tyr Gly Phe Leu Leu Pro Ala Arg Gln Ile Le - #u Pro Thr Ala Glu Glu            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - acc tgg ctt ggc ttg aag gca atc atg gag ca - #t gtg cga gac cac         ccc     1248                                                                     Thr Trp Leu Gly Leu Lys Ala Ile Met Glu Hi - #s Val Arg Asp His Pro                           405  - #               410  - #               415               - - tat                  - #                  - #                  - #                1251                                                                   Tyr                                                                             - -  - - <210> SEQ ID NO 4                                                    <211> LENGTH: 417                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Homo sapiens                                                    - - <400> SEQUENCE: 4                                                          - - Met Arg Leu Ile Leu Phe Phe Gly Ala Leu Ph - #e Gly His Ile Tyr Cys         1               5 - #                 10 - #                 15               - - Leu Glu Thr Phe Val Gly Asp Gln Val Leu Gl - #u Ile Val Pro Ser Asn                    20     - #             25     - #             30                   - - Glu Glu Gln Ile Lys Asn Leu Leu Gln Leu Gl - #u Ala Gln Glu His Leu                35         - #         40         - #         45                       - - Gln Leu Asp Phe Trp Lys Ser Pro Thr Thr Se - #r Gly Glu Thr Ala His            50             - #     55             - #     60                           - - Val Arg Val Pro Phe Val Asn Val Gln Ala Va - #l Lys Val Phe Leu Glu        65                 - # 70                 - # 75                 - # 80        - - Ser Gln Gly Ile Ala Tyr Ser Ile Met Ile Gl - #u Asp Val Gln Val Leu                        85 - #                 90 - #                 95               - - Leu Asp Lys Glu Asn Glu Glu Met Leu Phe As - #n Arg Arg Arg Glu Arg                   100      - #           105      - #           110                   - - Ser Gly Asn Phe Asn Phe Gly Ala Tyr His Th - #r Leu Glu Glu Ile Ser               115          - #       120          - #       125                       - - Gln Glu Met Asp Asn Leu Val Ala Glu His Pr - #o Gly Leu Val Ser Lys           130              - #   135              - #   140                           - - Val Asn Ile Gly Ser Ser Phe Glu Asn Arg Pr - #o Met Asn Val Leu Lys       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Phe Ser Thr Gly Gly Asp Lys Pro Ala Ile Tr - #p Leu Asp Ala Gly         Ile                                                                                              165  - #               170  - #               175              - - His Ala Arg Glu Trp Val Thr Gln Ala Thr Al - #a Leu Trp Thr Ala Asn                   180      - #           185      - #           190                   - - Lys Ile Val Ser Asp Tyr Gly Lys Asp Pro Se - #r Ile Thr Ser Ile Leu               195          - #       200          - #       205                       - - Asp Ala Leu Asp Ile Phe Leu Leu Pro Val Th - #r Asn Pro Asp Gly Tyr           210              - #   215              - #   220                           - - Val Phe Ser Gln Thr Lys Asn Arg Met Trp Ar - #g Lys Thr Arg Ser Lys       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Val Ser Gly Ser Leu Cys Val Gly Val Asp Pr - #o Asn Arg Asn Trp         Asp                                                                                              245  - #               250  - #               255              - - Ala Gly Phe Gly Gly Pro Gly Ala Ser Ser As - #n Pro Cys Ser Asp Ser                   260      - #           265      - #           270                   - - Tyr His Gly Pro Ser Ala Asn Ser Glu Val Gl - #u Val Lys Ser Ile Val               275          - #       280          - #       285                       - - Asp Phe Ile Lys Ser His Gly Lys Val Lys Al - #a Phe Ile Thr Leu His           290              - #   295              - #   300                           - - Ser Tyr Ser Gln Leu Leu Met Phe Pro Tyr Gl - #y Tyr Lys Cys Thr Lys       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Asp Asp Phe Asp Glu Leu Ser Glu Val Al - #a Gln Lys Ala Ala         Gln                                                                                              325  - #               330  - #               335              - - Ser Leu Arg Ser Leu His Gly Thr Lys Tyr Ly - #s Val Gly Pro Ile Cys                   340      - #           345      - #           350                   - - Ser Val Ile Tyr Gln Ala Ser Gly Gly Ser Il - #e Asp Trp Ser Tyr Asp               355          - #       360          - #       365                       - - Tyr Gly Ile Lys Tyr Ser Phe Ala Phe Glu Le - #u Arg Asp Thr Gly Arg           370              - #   375              - #   380                           - - Tyr Gly Phe Leu Leu Pro Ala Arg Gln Ile Le - #u Pro Thr Ala Glu Glu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Thr Trp Leu Gly Leu Lys Ala Ile Met Glu Hi - #s Val Arg Asp His         Pro                                                                                              405  - #               410  - #               415              - - Tyr                                                                        - -  - - <210> SEQ ID NO 5                                                    <211> LENGTH: 22                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence:nucleotide            primer from rat cDNA                                                      - - <400> SEQUENCE: 5                                                          - - cctgttattg gctgccctac tt           - #                  - #                      22                                                                       - -  - - <210> SEQ ID NO 6                                                    <211> LENGTH: 22                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer               sequence                                                                  - - <400> SEQUENCE: 6                                                          - - aagccaggtc tcttctgctg tc           - #                  - #                      22                                                                       - -  - - <210> SEQ ID NO 7                                                    <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer               sequence                                                                  - - <400> SEQUENCE: 7                                                          - - ggtccagtca gcagtgcttc c           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 8                                                    <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 8                                                          - - gagctcgaag gcgaaggagt a           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 9                                                    <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 9                                                          - - gagctcgaag ccgaaggagt a           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 10                                                   <211> LENGTH: 37                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 10                                                         - - tcccccgggc tgcaggatat cgatagctgg gctgtgt      - #                        - #      37                                                                       - -  - - <210> SEQ ID NO 11                                                   <211> LENGTH: 27                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer         - - <400> SEQUENCE: 11                                                         - - atcaagtact cctcccagct ccgggac          - #                  - #                  27                                                                       - -  - - <210> SEQ ID NO 12                                                   <211> LENGTH: 32                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 12                                                         - - tttatcaagc cagtggaggt attgactgga cc       - #                  - #               32                                                                       - -  - - <210> SEQ ID NO 13                                                   <211> LENGTH: 33                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 13                                                         - - aaggcaattt atcaaggcag tggaagcact att       - #                  - #              33                                                                       - -  - - <210> SEQ ID NO 14                                                   <211> LENGTH: 37                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer          - - <400> SEQUENCE: 14                                                         - - tcccccgggc tgcaggatat cgatagctgg gctgtgt      - #                        - #      37                                                                       - -  - - <210> SEQ ID NO 15                                                   <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer         - - <400> SEQUENCE: 15                                                         - - gctgaagctt cggaggactt t           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 16                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                             - - <400> SEQUENCE: 16                                                         - - tcttgaccgc ctggatgctg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 17                                                   <211> LENGTH: 419                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Homo sapiens                                                    - - <400> SEQUENCE: 17                                                         - - Met Arg Gly Leu Leu Val Leu Ser Val Leu Le - #u Gly Ala Val Phe Gly         1               5 - #                 10 - #                 15               - - Lys Glu Asp Phe Val Gly His Gln Val Leu Ar - #g Ile Ser Val Ala Asp                    20     - #             25     - #             30                   - - Glu Ala Gln Val Gln Lys Val Lys Glu Leu Gl - #u Asp Leu Glu His Leu                35         - #         40         - #         45                       - - Gln Leu Asp Phe Trp Arg Gly Pro Ala His Pr - #o Gly Ser Pro Ile Asp            50             - #     55             - #     60                           - - Val Arg Met Pro Phe Pro Ser Ile Gln Ala Va - #l Lys Ile Phe Leu Glu        65                 - # 70                 - # 75                 - # 80        - - Ser His Gly Ile Ser Tyr Glu Thr Met Ile Gl - #u Asp Val Gln Ser Leu                        85 - #                 90 - #                 95               - - Leu Asp Glu Glu Gln Glu Gln Met Phe Ala Ph - #e Arg Ser Arg Ala Arg                   100      - #           105      - #           110                   - - Ser Thr Asp Thr Phe Asn Tyr Ala Thr Tyr Hi - #s Thr Leu Glu Glu Ile               115          - #       120          - #       125                       - - Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu As - #n Pro His Leu Val Ser           130              - #   135              - #   140                           - - Lys Ile Gln Ile Gly Asn Thr Tyr Glu Gly Ar - #g Pro Ile Tyr Val Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Lys Phe Ser Thr Gly Gly Ser Lys Arg Pro Al - #a Ile Trp Ile Asp         Thr                                                                                              165  - #               170  - #               175              - - Gly Ile His Ser Arg Glu Trp Val Thr Gln Al - #a Ser Gly Val Trp Phe                   180      - #           185      - #           190                   - - Ala Lys Lys Ile Thr Gln Asp Tyr Gly Gln As - #p Ala Ala Phe Thr Ala               195          - #       200          - #       205                       - - Ile Leu Asp Thr Leu Asp Ile Phe Leu Glu Il - #e Val Thr Asn Pro Asp           210              - #   215              - #   220                           - - Gly Phe Ala Phe Thr His Ser Thr Asn Arg Me - #t Trp Arg Lys Thr Arg       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ser His Thr Ala Gly Ser Leu Cys Ile Gly Va - #l Asp Pro Asn Arg         Asn                                                                                              245  - #               250  - #               255              - - Trp Asp Ala Gly Phe Gly Leu Ser Gly Ala Se - #r Ser Asn Pro Cys Ser                   260      - #           265      - #           270                   - - Glu Thr Tyr His Gly Lys Phe Ala Asn Ser Gl - #u Val Glu Val Lys Ser               275          - #       280          - #       285                       - - Ile Val Asp Phe Val Lys Asp His Gly Asn Il - #e Lys Ala Phe Ile Ser           290              - #   295              - #   300                           - - Ile His Ser Tyr Ser Gln Leu Leu Met Tyr Pr - #o Tyr Gly Tyr Lys Thr       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Glu Pro Val Pro Asp Gln Asp Glu Leu Asp Gl - #n Leu Ser Lys Ala         Ala                                                                                              325  - #               330  - #               335              - - Val Thr Ala Leu Ala Ser Leu Tyr Gly Thr Ly - #s Phe Asn Tyr Gly Ser                   340      - #           345      - #           350                   - - Ile Ile Lys Ala Ile Tyr Gln Ala Ser Gly Se - #r Thr Ile Asp Trp Thr               355          - #       360          - #       365                       - - Tyr Ser Gln Gly Ile Lys Tyr Ser Phe Gly Ph - #e Glu Leu Arg Asp Thr           370              - #   375              - #   380                           - - Gly Arg Tyr Gly Phe Leu Leu Pro Ala Ser Gl - #n Ile Ile Pro Thr Ala       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Lys Glu Thr Trp Leu Ala Leu Leu Thr Ile Me - #t Glu His Thr Leu         Asn                                                                                              405  - #               410  - #               415              - - His Pro Tyr                                                                - -  - - <210> SEQ ID NO 18                                                   <211> LENGTH: 417                                                              <212> TYPE: PRT                                                                <213> ORGANISM: Homo sapiens                                                    - - <400> SEQUENCE: 18                                                         - - Met Arg Leu Ile Leu Phe Phe Gly Ala Leu Ph - #e Gly His Ile Tyr Cys         1               5 - #                 10 - #                 15               - - Leu Glu Thr Phe Val Gly Asp Gln Val Leu Gl - #u Ile Val Pro Ser Asn                    20     - #             25     - #             30                   - - Glu Glu Gln Ile Lys Asn Leu Leu Gln Leu Gl - #u Ala Gln Glu His Leu                35         - #         40         - #         45                       - - Gln Leu Asp Phe Trp Lys Ser Pro Thr Thr Se - #r Gly Glu Thr Ala His            50             - #     55             - #     60                           - - Val Arg Val Pro Phe Val Asn Val Gln Ala Va - #l Lys Val Phe Leu Glu        65                 - # 70                 - # 75                 - # 80        - - Ser Gln Gly Ile Ala Tyr Ser Ile Met Ile Gl - #u Asp Val Gln Val Leu                        85 - #                 90 - #                 95               - - Leu Asp Lys Glu Asn Glu Glu Met Leu Phe As - #n Arg Arg Arg Glu Arg                   100      - #           105      - #           110                   - - Ser Gly Asn Phe Asn Phe Gly Ala Tyr His Th - #r Leu Glu Glu Ile Ser               115          - #       120          - #       125                       - - Gln Glu Met Asp Asn Leu Val Ala Glu His Pr - #o Gly Leu Val Ser Lys           130              - #   135              - #   140                           - - Val Asn Ile Gly Ser Ser Phe Glu Asn Arg Pr - #o Met Asn Val Leu Lys       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Phe Ser Thr Gly Gly Asp Lys Pro Ala Ile Tr - #p Leu Asp Ala Gly         Ile                                                                                              165  - #               170  - #               175              - - His Ala Arg Glu Trp Val Thr Gln Ala Thr Al - #a Leu Trp Thr Ala Asn                   180      - #           185      - #           190                   - - Lys Ile Val Ser Asp Tyr Gly Lys Asp Pro Se - #r Ile Thr Ser Ile Leu               195          - #       200          - #       205                       - - Asp Ala Leu Asp Ile Phe Leu Leu Pro Val Th - #r Asn Pro Asp Gly Tyr           210              - #   215              - #   220                           - - Val Phe Ser Gln Thr Lys Asn Arg Met Trp Ar - #g Lys Thr Arg Ser Lys       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Val Ser Gly Ser Leu Cys Val Gly Val Asp Pr - #o Asn Arg Asn Trp         Asp                                                                                              245  - #               250  - #               255              - - Ala Gly Phe Gly Gly Pro Gly Ala Ser Ser As - #n Pro Cys Ser Asp Ser                   260      - #           265      - #           270                   - - Tyr His Gly Pro Ser Ala Asn Ser Glu Val Gl - #u Val Lys Ser Ile Val               275          - #       280          - #       285                       - - Asp Phe Ile Lys Ser His Gly Lys Val Lys Al - #a Phe Ile Thr Leu His           290              - #   295              - #   300                           - - Ser Tyr Ser Gln Leu Leu Met Phe Pro Tyr Gl - #y Tyr Lys Cys Thr Lys       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Asp Asp Phe Asp Glu Leu Ser Glu Val Al - #a Gln Lys Ala Ala         Gln                                                                                              325  - #               330  - #               335              - - Ser Leu Arg Ser Leu His Gly Thr Lys Tyr Ly - #s Val Gly Pro Ile Cys                   340      - #           345      - #           350                   - - Ser Val Ile Tyr Gln Ala Ser Gly Gly Ser Il - #e Asp Trp Ser Tyr Asp               355          - #       360          - #       365                       - - Tyr Gly Ile Lys Tyr Ser Phe Gly Phe Glu Le - #u Arg Asp Thr Gly Arg           370              - #   375              - #   380                           - - Tyr Gly Phe Leu Leu Pro Ala Arg Gln Ile Le - #u Pro Thr Ala Glu Glu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Thr Trp Leu Gly Leu Lys Ala Ile Met Glu Hi - #s Val Arg Asp His         Pro                                                                                              405  - #               410  - #               415              - - Tyr                                                                        - -  - - <210> SEQ ID NO 19                                                   <211> LENGTH: 50                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer               sequence                                                                  - - <400> SEQUENCE: 19                                                         - - gcagaagctt cagaaacgtt tgtgggagat caagttcttg agattgtacc  - #                   50                                                                          - -  - - <210> SEQ ID NO 20                                                   <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer               sequence                                                                  - - <400> SEQUENCE: 20                                                         - - ctctttgtcc aacaggacct g           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 21                                                   <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer               sequence                                                                  - - <400> SEQUENCE: 21                                                         - - tcctccactg ccttggtaga t           - #                  - #                       - #21                                                                    - -  - - <210> SEQ ID NO 22                                                   <211> LENGTH: 21                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer               sequence                                                                  - - <400> SEQUENCE: 22                                                         - - cagttcaaag ccaaatgagt a           - #                  - #                       - #21                                                                  __________________________________________________________________________ 

We claim:
 1. A conjugate comprising a cell targeting molecule and a mutant human carboxypeptidase A enzyme wherein the enzyme possesses amino acid substitutions at one or more of residues 203, 210, 242, 244, 250, 253, 255, 267, 268, 269 and
 305. 2. A conjugate according to claim 1 wherein the amino acid substitutions are selected from the following:Gly at 250 and 268, Gly at 253 and 268, Gly at 250 and His at 268, Gly at 250, Ala at 255 and His 268, His at 268, and Gly at
 268. 3. A conjugate according to claim 2 wherein the targetting molecule is one of an antibody, a hormone, a ligand, a cytokine, an antigen, an oligonucleotide and a peptidomimetic.
 4. A conjugate according to claim 2 wherein the substitution is Gly at
 268. 5. A conjugate according to claim 4 wherein the targetting molecule is one of an antibody, a hormone, a ligand, a cytokine, an antigen, an oligonucleotide and a peptidomimetic.
 6. A conjugate according to claim 1 wherein the targetting molecule is one of an antibody, a hormone, a ligand, a cytokine, an antigen, an oligonucleotide and a peptidomimetic.
 7. A conjugate according to claim 6 wherein the antibody is a polyclonal antibody, a monoclonal antibody or a fragment thereof.
 8. A conjugate according to claim 7 wherein the antibody of fragment thereof is humanised.
 9. A conjugate according to claim 6, wherein the targeting molecule is joined to the enzyme by a linker molecule.
 10. An enzyme comprising a mutant human caboxypeptidase A enzyme wherein the enzyme possesses amino acid substitutions at one or more of residues 203, 210, 242, 244, 250, 253, 255, 267, 268, 269 and
 305. 11. An enzyme according to claim 10 wherein the amino acid substitutions are selected from the following:Gly at 250 and 268, Gly at 253 and 268, Gly at 250 and His at 268, Gly at 250, Ala at 255 and His 268, His at 268, and Gly at
 268. 12. An enzyme according to claim 11 wherein the substitution is Gly at
 268. 